The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

A 4D-microscopic analysis of the germ band in the isopod crustacean Porcellio scaber (Malacostraca, Peracarida)—developmental and phylogenetic implications

Malacostracan crustaceans have evolved a conserved stereotyped cell division pattern in the post-naupliar germ band. This cleavage pattern is unique in arthropods investigated so far, and allows a combined analysis of gene expression and cell lineage during segmentation and organ development at the level of individual cells. To investigate the cell lineage in the germ band of the isopod Porcellio scaber, we used a 4D-microscopy system, which enables us to analyse every cell event in the living embryo. The study was combined with the analysis of the expression of the gene engrailed (en) at different stages of germ band formation. Our findings confirm the results of earlier investigations of the cell division pattern in the posterior part of the isopod germ band. Furthermore, we can show that in the anterior region, in contrast to the posterior part, cleavage directions are variable and cell sorting takes place—similar to other arthropod germ bands. Additionally, the gene expression pattern of en in this region is not as regular as in the post-naupliar germ band, and only later becomes regulated into its characteristic stripe pattern. The comparison of the cell lineage of P. scaber with that of other malacostracan crustaceans shows an enhancement in the velocity of cell divisions relative to the arrangement of these cells in rows in the isopod germ band. The striking similarity of the formation of the genealogical units in the anterior part suggests a sister group relationship between the peracarid taxa Tanaidacea and Isopoda.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous,endocrine and plicated cells

Summary The following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.The authors are grateful to Mr. G. Tognon for technical help and to the Staff of the Stazione Idrobiologica di Chioggia for their assistance in collecting material. Work supported by a C.N.R. Grant from the Istituto di Biologia del Mare, Venezia, Contract n. 71.00396/04.115.542.     

The ultrastructure of cellular division (autosporogenesis) in the coccoid green alga,Trebouxia aggregata,revealed by rapid freeze fixation and freeze substitution

Summary Adequate ultrastructural preservation of cells of the green algaTrebouxia aggregata is achieved by immersion freeze fixation using liquid propane followed by freeze substitution and resin embedding at ambient temperature. Despite differential staining of membranes, using this method we have been able to study plasma membrane biogenesis during cellular division. Daughter protoplasts are separated by an ingrowing septum of plasma membrane that extends into the cell from a particular site at the peripheral plasma membrane marked by centrioles. Septum development involves tip growth followed by lateral growth. This growth seems to involve transfer of membrane from an adjacent partially coated reticulum to the septum plasma membrane. The reticulum which extends from nearby Golgi stacks to the area of septum growth is associated with an extensive array of microtubules. After daughter protoplasts are completely separated, each one becomes surrounded by a cell wall which is distinct from the persisting mother wall. The ultrastructural evidence suggests that cells ofT. aggregata are autospores rather than vegetative cells.Abbreviations C centriole - ER endoplasmic reticulum - G Golgi body - MTOC microtubule organizing center - Mt(s) microtubule(s) - N nucleus - P primary septum - PCR partially coated reticulum - PM plasma membrane - Py pyrenoid - S septum     

Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster

Summary An ultrastructural study was made of the ring canal system which connects the sister ovarian cystocytes that arise in the germaria of wild type Drosophila melanogaster females. It was discovered that during an oogonial mitosis both chromosomes and spindle are enclosed by a multilayered, perforated membrane system derived (at least in part) from the nuclear envelope. The cytokinesis of stem line oogonia takes place through the formation of a cleavage furrow. A second method of formation of plasma membrane is found in the case of cystocytes. It involves the production along the plane of division of a plaque of interconnected vesicles and tubules and later the coalescence of nearby tubules to form continuous sheets of membrane which segregate the cytoplasms of the sister cells. However, these remain connected by a canal which is enclosed by a ring-shaped rim that is completed prior to the plasma membrane to which the rim is subsequently attached. It is postulated that the rim represents a transformed midbody. As development proceeds the canal becomes wider, its rim becomes thicker, and the inner circumference of the rim becomes coated with a thick deposit having different cytochemical properties than the rim itself. Cystocyte divisions produce sister cells which differ in that one receives all previously formed canals; the other none. In the case of the last division (and perhaps in earlier ones as well) the sister cell receiving all previously formed canals also receives more cytoplasm than its sister. As the cells of the cluster grow, the canals remain close together. This finding suggests that when new plasma membrane is synthesized, it is added in areas remote from the canals. An investigation of the positioning in three dimensions of the fifteen canals of a newly formed, 16 cellcluster suggests that the spindles produced at one division are never parallel to those formed at the subsequent division. This continual shifting of the axes of the spindles at consecutive divisions presumably results in the branching chains of cells which characterize a cystocyte cluster. The possession of a unique pattern of cortical structures by two cystocytes is accompanied by the nuclear synthesis of synaptonemal complexes. The other fourteen cystocytes differentiate into nurse cells. In the most posterior portion of the germarium one of the two potential oocytes switches to the nurse cell developmental pathway. This switched off oocyte and the definitive oocyte grow at rates which differ greatly and are correlated to the amount of contact between their surfaces and certain follicle cells. As development proceeds centrioles accumulate in the oocyte, and most of these are thought to have been carried from the nurse cells into the oocyte in the nutrient stream.The authors are grateful to Richard Z. Belch and James E. Bradof for their conscientious assistance and to E. John Pfiffner for preparation of the inked drawings and construction of the Polyform models. This research was supported by the National Science Foundation grant GB7457.     

Ultrastructure of vegetative organization and cell division in the freshwater red algaCompsopogon

Summary Uniseriate filaments of the freshwater red algaCompsopogon coeruleus were examined by transmission electron microscopy for details of vegetative organization and cell division with the goal of providing useful taxonomic characters. Each cell's single, complex chloroplast contains a peripheral encircling thylakoid, and unlike the vast majority of red algae, the cis-regions of dictyosomes are not consistently juxtaposed with mitochondria. These subcellular features, which are present in all examined genera in theCompsopogonales, Erythropeltidales, andRhodochaetales, along with certain unique reproductive characteristics, unify these three orders. During mitosis in uncorticated axial cells, a small, ring-shaped nucleus associated organelle (NAO) is located at each division pole, an intranuclear spindle comes to a moderately acute focus at the flattened, fenestrated metaphase-anaphase division poles and perinuclear ER partially encloses dividing nuclei, including a well-developed interzonal midpiece. The cleavage furrow penetrates the large, central vacuolar region to separate daughter nuclei. These cell division features most closely resemble the pattern described for the orderCeramiales. Our observations of vegetative and dividing cells ofC. coeruleus supplement the growing volume of evidence in favour of uniting all red algae into a single class without subclass designations.Abbreviations ER endoplasmic reticulum - IZM interzonal midpiece - MT microtubule - MTOC microtubule organizing center - NAO nucleus associated organelle - NE nuclear envelope - PER perinuclear endoplasmic reticulum     

OBSERVATIONS ON THE STRUCTURE OF SOME FORMS OF GOMPHONEMA PARVULUM KÜTZ. III. FRUSTULE FORMATION1

Gomphonema parvulum Kütz. was investigated by electron microscopy for details of frustule formation. An expansion of the cell along the pervalvar plane occurs prior to cell division. After nuclear division the organelles are, separated into 2 entities, either by division or by dispersion. The cell divides into 2 halves by the invagination of the plasmalemma which is derived from Golgi vesicular activity. When cytoplasmic cleavage, is complete, the Golgi actively produces electronlucent vesicles which collect and coalesce beneath the. plasmalemma to form the silicalemma around the silicon deposition vesicle. The endoplasmic reticulum is also closely associated with this vesicular activity. The vesicle gradually expands and becomes extremely electron dense as silica is deposited within it—first in the region, followed by the mantle edge. When the valve is mature, Golgi vesicles collect and fuse to form the silicalemma of the first girdle band. The first girdle band becomes aligned against the mantle edge on completion, by the “sloughing off” of the external silicalemma and plasmalemma. The second and third bands are formed, individually in a similar manner. Separation of the 2 daughter cells commences at the apical pole and progresses to the basal pole. The plasmalemma and external silicalemma are “sloughed off” so that the 2 cells can separate. The inner segment of the silicalemma becomes the new plasmalemma of the daughter cell.     

A further contribution to the study of the contact region of Octopus synaptosomes

Summary Octopus synaptosomes have been examined after glutaraldehyde fixation and phosphotungstic acid (PTA) staining of non-osmicated tissue. The results concentrate on the appearance of the contact region between the presynaptic component of synaptosomes and their postsynaptic processes. Membranes have a triple-layered appearance, consisting of an electronopaque internal coat, an electrontranslucent band and an electronopaque external coat. Good examples of this are found in synaptosomal, dendritic and axonal membranes. At specialized synaptic contact regions the external coats of the pre- and postsynaptic membranes coalesce to form a prominent synaptic plate, which has a width of 18 nm and is subdivided into zones of varying electronopacity. It is suggested that this plate is formed from the specialized external coat of the postsynaptic membrane and the unspecialized external coat of the presynaptic membrane. Presynaptic spicules extend from the internal coat of the presynaptic membrane. They are closely associated with elements of the presynaptic network.It is suggested that the synaptic plate is probably composed of mucopolysaccharides, while the relation of the plate to acetylcholinesterase is discussed. It is proposed that functional localization at the synapse is less precise in octopus than vertebrates.I would like to express my thanks to Professors J. Z. Young, F. R. S. and E. G. Gray for their helpful advice, and also Mr. S. Waterman for photographic assistance.     

Microspore ofSecale cereale as a transfer cell type

Summary In the mature microspore ofSecale cereale, a set of wall ingrowths deposited as the first (outer) intine layer between exine and the microspore plasma membrane, are revealed by electron microscopy. The wall ingrowths form a girdle in the vicinity of the apertural region at the external pole of microspore which is in contact with the tapetum, so the microspore can be considered as a transfer cell which is polarized. After microspore division the second (inner) intine layer is deposited by the vegetative cell and forms a labyrinth of branched wall ingrowths. As a result, the periphery of a vegetative cell is also irregular and appears as very thin plasmatubules or evaginations delimited by plasma membrane and penetrating the pollen wall.The possible functions of the microspore as a transfer cell and the wall-membrane system of the vegetative cell are discussed.     

A correlative study of experimentally changed first cleavage and Janus development in the trunk of Platynereis dumerilii (Annelida,Polychaeta)

Summary Among zygotes of Platynereis dumerilii treated with cytochalasin B (CCB) prior to first cleavage, a wide variety of developmental effects were observed. One effect is a delay in the first cleavage. Treated embryos may skip the first or even more than one cleavage cycle and become multinucleated. Once these eggs start cleaving their cleavage plane takes the same position as in synchronously fertilized controls. Accordingly, the first cleavage in embryos having skipped the first normal cleavage cycle is meridional and equal, but their second cleavage is equatorial as in the third cleavage in controls. None of the embryos that were observed to skip early cleavages showed normal organogenesis, but developed into vesicle-shaped embryos with little cytological differentiation. Another effect of CCB treatment is altered blastomere size in those embryos which begin cleaving in synchrony with controls. While the majority of treated embryos followed a normal cleavage pattern, i.e. they cleaved at the right time and inequally, some of them cleaved equally or almost equally (adequally). Most of these embryos showed cleavage defects in subsequent cleavage cycles and became abnormal vesicle-shaped embryos. However, some of these embryos cleaving on schedule and equally or adequally developed into juvenile worms showing complete duplication of urites and parapodial rows (0.3% of all treated eggs) and are described as Janus duplicitates. This means that the occurrence of duplicitates and geometrically altered first cleavage patterns are correlated phenomena. The character and origin of the duplications and the consequences for dorsoventral polarity are discussed.     

The F-actin distribution during microsporogenesis in Magnolia soulangeana Soul. (Magnoliaceae)

Summary F-actin distribution during male meiosis in Magnolia soulangeana was studied by means of fluorescence microscopy following staining with rhodaminephalloidin. Actin filaments were observed to persist during all of the developmental stages of meiosis. Four main types of configurations were recognized: (1) peripheral filaments underlying the plasma membrane (cortical network); (2) filaments dispersed throughout the inner cytoplasm (central cytoplasmic network); (3) filaments associated with the meiotic spindles; (4) filaments associated with the phragmoplasts. The cortical and central cytoplasmic filaments exhibited different behaviours. Whereas the cortical network remained present in an apparently unchanged form during all of the meiotic stages, the central cytoplasmic filaments, although they never completely disappeared, were reduced and concentrated around the nucleus at the end of prophase. At metaphase, fluorescent spindles consisting of filament bundles running from pole to pole or being interrupted at the equatorial zone could be seen. At the end of both the first and second division of meiosis, fluorescent bands of filaments (disks) appeared at the level of the cell division planes (equatorial regions) where cleavage furrows were constituted. These cleavage furrows did not form when floral buds were cultivated in a cytochalasin-containing medium. Our results show that during microsporogenesis in M. soulangeana the actin filaments constitute a highly complex and dynamic system that is involved in particular in cytoplasm cleavage of the meiocytes.     

Mitosis and cytokinesis inTribonema regulare (Tribophyceae,Chrysophyta)

Summary The ultrastructure of mitosis and cytokinesis of the uninucleateTribonema regulare has been investigated by employing transmission electron microscopy. Prophase is characterized by settlement of a pair of centrioles at the presumptive poles of the spindle, metaphase by equatorial bulging of the nucleus, anaphase by non-synchronous separation of the chromosomes, and telophase by a persistent, strongly elongated, interzonal spindle. Throughout mitosis, at each pole dictyosomes are associated with the polar gaps of the nuclear envelope that otherwise remains intact. Cytokinesis does not immediately follow mitosis; from the static images it can be concluded that it is necessary for the daughter nuclei to approach each other before cytokinesis is initiated by complete division of the protoplast via plasma membrane cleavage. Afterwards, a ring of cell wall material is deposited close near the lateral wall in the plane of protoplast separation followed by a simultaneous or centripetal development of a single integral partitioning septum. Once the septum is completed, the cylindrical portion of the H-shaped segment is manufactured. The phylogenetic position ofTribonema amongst those algae, which may have evolved from unicells into filaments, is discussed.     

Grillotia australis n. sp. and G. pristiophori n. sp. (Cestoda: Trypanorhyncha) from Australian elasmobranch and teleost fishes

Two new species of Grillotia are described from elasmobranch and teleost fishes from south-eastern Australia. G. australis n. sp., from the Australian angel shark Squatina australis. Regan, most closely resembles G. smarisgora (Wagener, 1854) and G. angeli Dollfus, 1969, differing from both species in the presence of smaller bulbs, two or occasionally three hooks in each intercalary row in the basal region, reduced to one in the metabasal region compared with four or five hooks in the metabasal region of G. smarisgora and a single hook in G. angeli, and in the limited extent of the band of hooklets on the external surface in the basal region of the tentacle, a region which is covered with hooks in G. smarisgora. Plerocerci of this species were found in the mackerel Trachurus declivis (Jenys) (site not known) from Tasmania. G. pristiophori n. sp., from the saw sharks Pristiophorus cirratus (Latham) and P. nudipinnis Günther, most closely resembles G. spinosissima Dollfus, 1969 in possessing a scolex covered with spiniform microtriches, but differs in having six rather than five hooks in each principal row, no intercalary hooks and by possessing a band of hooklets on the external surface of the tentacle which diminishes distally into a single file, rather than persisting as a band eight to nine files wide. G. pristiophori is the first trypanorhynch to be recorded from saw-sharks.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Asynchronous mitotic domains during blastoderm formation inMusca domestica L. (Diptera)

Summary We describe the mitotic cleavage patterns during blastoderm stage of the house flyMusca domestica L. Nuclear divisions up to mitotic stage 11 are apparently synchronous. Beginning with stage 12, nuclear divisions in the posterior third of the embryo lag behind, resulting first in a parasynchronous and finally in an asynchronous cleavage pattern. Thus a stage exists where all nuclei in the anterior region have completed 14 nuclear division cycles, while those in the posterior region have completed only 13 cycles. The border region between these nuclei is well defined and lies at 35% EL (egg length), the expression border of a gap gene. This border region is about 4–5 nuclei wide and shows a specialized mitotic behaviour.     

Division polarity,development and configuration of microtubule arrays in bryophyte meiosis

Summary First and second division spindles and the three cell plates of moss meiosis are oriented in accordance with polarity established during meiotic prophase. Plastids are located at the second division poles and cytoplasmic infurrowing marks the planes along which the cytoplasm will cleave into four spores. Anaphase I spindles that terminate in two focal points of microtubules straddling opposite cleavage furrows reflect the unusual tetrahedral origin of the functionally bipolar spindle. The organelles (except for the plastids which remain in the four cytoplasmic lobes) are polarized in the first division equatorial region at the time of phragmoplast microtubule assembly and remain in a distinct band after microtubule disassembly. Prophasic spindles appear to be directly transformed into metaphase II spindles in the predetermined axes between mutually perpendicular pairs of plastids. Cell plates form by vesicle coalescence in the equatorial regions of the two sets of second division phragmoplasts at approximately the same time as a cell plate belatedly forms in the organelle band. The cytoplasmic markers (plastid migration, cytoplasmic lobing and infurrowing) that predict poles and cleavage planes in free cells lacking a preprophase band strongly strengthens the concept that division sites are capable of preserving preprogrammed signals that can be triggered later in the process of cell division.     

The fine structure of the carapace integument of Daphnia magna straus (Crustacea branchiopoda)

Summary The structure of the two integumental layers comprising the carapace of female D. magna was examined at several points through the molt cycle. The epicuticle and procuticle are simple in organisation; pore canals are absent but intracuticular fibres are present, forming complexes with invaginations of the epidermal plasma membrane similar to such complexes described in the literature for other arthropods. The epidermis consists almost entirely of cuticle-secreting cells. Secretion of the new cuticle begins when 50–67% of the instar has elapsed by which time the epidermal cells have increased in height and their nuclei have become more rounded. However, other presumed secretory phenomena observed viz. the formation of dense core vesicles by Golgi bodies, and the occurrence of these and coated vesicles near the apical plasma membrane are not restricted to any particular period during the molt cycle. This suggests that the mechanisms of cuticle secretion do not undergo marked changes in activity as they do in decapods; presumably this relative continuity is related to the much shorter molt cycle of cladocerans.The technical assistance of G.A. Bance, and the financial support provided by the National Research Council of Canada are gratefully acknowledged     

The microtubule cytoskeleton in developing cysts of the green algaAcetabularia: Involvement in cell wall differentiation

Summary Cysts of the green algaAcetabularia develop a unique lid structure to enable the release of gametes. This lid is separated from the rest of the thick cellulose cell wall by a circular fault line formed within the fibrillar texture of the wall. By immunofluorescence microscopy, we show that, prior to the first division of the single cyst nucleus, the radially symmetrical, perinuclear microtubule system which is a remnant carried over from previous developmental stages of cyst morphogenesis transforms into a circular microtubule band (CMB) around the nucleus. This band consisting of only a few bundled microtubules beneath the plasma membrane encircles the cyst nucleus at a distance of 75 to 100m. In a previous fine structural study, a lid-forming apparatus (LFA) was described as a circular band of rod-like structures in the plane of the plasma membrane, demarcating the contour of the future lid. Both the CMB and the LFA are superimposed on the rim of the lid. We therefore propose that the microtubule band is a component of the LFA identical with the rod-like structures. Formation of the CMB and, hence, lid formation are blocked by the microtubule-specific herbicide Oryzalin but not by the actin filament-disrupting inhibitor cytochalasin D. Upon recovery from Oryzalin treatment, the nuclei but not the prospective sites of the CMBs serve as nucleation centers, indicating that the CMB is not formed by a pre-existing template in the plasma membrane. This suggests that the dynamic behavior of the microtubules within the perinuclear microtubule cytoskeleton gives rise to the CMB. Since the stage of CMB assembly marks the beginning of cell wall formation, it is proposed that the CMB determines the position of the lid by spatially controlling cell wall deposition. On the basis of current hypotheses, two scenarios for the role of the LFA/CMB in lid formation are discussed.Abbreviations CMB circular microtubule band - EGTA ethylene glycol bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - LFA lid-forming-apparatus - MAP microtubule-associated protein - MT microtubule - MTOC microtubuleorganizing center Dedicated to the memory of Professor Oswald Kiermayer