hESC derived neuro-epithelial rosettes recapitulate early mammalian neurulation events; an in vitro model

The in vitro neuralization of hESCs has been widely used to generate central and peripheral nervous system components from neural precursors (Bajpai et al., 2009; Curchoe et al., 2010), most often through an intermediate “rosette” stage. Here we confirm that hESC derived neuro-epithelial rosettes express many characteristics of the developing embryonic neural plate (Aaku-Saraste et al., 1996), characterized by expression of the tight junction proteins ZO-1 and N-Cadherin. Moreover, neuro-epithelial rosettes display a characteristic acetylated alpha tubulin cytoskeletal arrangement (similar to that observed in the developing embryonic neural plate) (Bhattacharyya et al., 1994).Demonstrated here for the first time MKLP was observed in a hESC model system. We found MKLP expression in small particles in between mitotic spindles, large particles aggregating in the lumen of neuroepithelial rosettes, and we did not observe MKLP in the nucleus of hESC derived neural precursors as previously described in the HeLa cell line. We observed MKLP + particles in aggregations in the lumen of “early” rosette structures. Furthermore, we observed that MKLP⁺ particle aggregations can also be lost from the lumens of hESC derived neuro-epithelial rosettes, similar to a phenomenon observed in the developing neural tube in vivo (Marzesco et al., 2005). We determined that this loss of MKLP⁺ particles occurs from “late” as opposed to “early” stage neuro-epithelial rosettes (characterized by junction type).Disrupting the apical-basal polarization of “early” stage rosettes with a 1% Matrigel overlay (Krtolica et al., 2007) nearly ablates MKLP particle aggregation in the lumen of rosettes, demonstrating that the apical-basal polarity of early NE cells is necessary for lumenal MKLP particle aggregation.We conclude that early hESC derived neuro-epithelial rosettes can model early neurulation events, such as the transition from neural plate like cells to neural tube like cells (i.e. symmetric to asymmetric NE cell division) demonstrated by polarized MKLP particle inheritance and distribution using junction type as a measure of stage.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Arrays of plasma-membrane “rosettes” involved in cellulose microfibril formation of Spirogyra

The cell-wall structure and plasma-membrane particle arrangement during cell wall formation of the filamentous chlorophycean alga Spirogyra sp. was investigated with the freeze-fracture technique. The cell wall consists of a thick outer slime layer and a multilayered inner wall with ribbon-like microfibrils. This inner wall shows three differing orientations of microfibrils: random orientation on its outside, followed by axial bundles of parallel microfibrils, and several internal layers of bands of mostly five to six parallel associated microfibrils with transverse to oblique orientation. The extraplasmatic fracture face of the plasma membrane shows microfibril imprints, relatively few particles, and “terminal complexes” arranged in a hexagonal package at the end of the imprint of a microfibril band. The plasmatic fracture face of the plasma membrane is rich in particles. In places, it reveals hexagonal arrays of “rosettes”. These rosettes are best demonstrable with the double-replica technique. These findings on rosette arrays of the zygnematacean alga Spirogyra are compared in detail with the published data on the desmidiacean algae Micrasterias and Closterium.     

Properties of DNA rosettes and their relevance to chromosome structure

We have studied the spreading conditions that lead to the formation of rosettes in DNA and chromatin preparations from the amphibians Bufo marinus and Bolitoglossa subpalmata and the bacterium Shigella. Both nuclear preparations and extensively deproteinized DNA produced rosettes. The longest fibers and the most symmetric rosettes were observed in amphibian nuclear spreadings. In this procedure purified nuclei were submitted immediately to Kleinschmidt spreading over various types of hypophase. Distilled-water hypophases were most conducive for rosette production or stability. Rosettes were observed with cytochrome C as the basic protein, but not with ribonuclease A and bovine serum albumin. We cannot prove that all rosettes are artifacts of the spreading procedure, but we believe that at least some result from the expansion of compact DNA doughnuts and other structures that are apparently formed in the presence of basic proteins in salt concentrations over 40 mM (Olins and Olins 1971; Manning 1979). The dilute hypophase requirement is explainable by the assumption that dilution and spreading effects unfold a compact precursor. Occasionally we have detected structures that appear to be intermediates in the process of doughnut unfolding and that illustrate a procedure that may give rise to rosettes.     

Prediction of signal recognition particle RNA genes

We describe a method for prediction of genes that encode the RNA component of the signal recognition particle (SRP). A heuristic search for the strongly conserved helix 8 motif of SRP RNA is combined with covariance models that are based on previously known SRP RNA sequences. By screening available genomic sequences we have identified a large number of novel SRP RNA genes and we can account for at least one gene in every genome that has been completely sequenced. Novel bacterial RNAs include that of Thermotoga maritima, which, unlike all other non-gram-positive eubacteria, is predicted to have an Alu domain. We have also found the RNAs of Lactococcus lactis and Staphylococcus to have an unusual UGAC tetraloop in helix 8 instead of the normal GNRA sequence. An investigation of yeast RNAs reveals conserved sequence elements of the Alu domain that aid in the analysis of these RNAs. Analysis of the human genome reveals only two likely genes, both on chromosome 14. Our method for SRP RNA gene prediction is the first convenient tool for this task and should be useful in genome annotation.     

Intramembranous particles in the form of ridges,bracelets or assemblies in arthropod tissues

Non-junctional intramembranous particle arrays in the form of ridges, bracelets or rectilinear assemblies have been found by freeze-fracturingin the cytoplasmic half or P face of the plasma membrane in a variety of arthropod tissues. These tissues include both excitable cells, nerve and muscle, and such other cells as those from the intestinal tract, the tracheal system and the connective tissue. The intramembranous ridges are short rows of fused particles about 10 nm in diameter; comparable particles comprise the bracelets and the rectilinear aggregates, although the former are of lower profile. In cells sending out cytoplasmic projections during migration and development, for example, axons in embryonic, newly hatched or pupal tissues, tracheoles or fibroblasts, the intramembranous ridges are always aligned parallel to the longitudinal axis of the cellular process. The physiological significance of these may be that they play some role in recognition during development, perhaps by contact guidance. The ridges and rectilinear arrays found in the gut could also be involved in recognition and/or adhesion. In muscle, bead-like ridges are intimately associated with the transverse tubular system and may have a receptor function. Irregular and circular low-profile ridges occur in the tissues of the horseshoe crab, Limulus, and ‘bracelet’ forms are found in the inner membrane of insect pupal tracheae. The latter may play a part in the initiation and development of small tracheoles.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Morphological Heterogeneity and Attachment of Phaeobacter inhibens

The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.     

Shell structure in Difflugia Lobostoma observed by scanning and transmission electron microscopy

Observations by scanning and transmission electron microscopy provide information about shells of Difflugia lobostoma which suggests a complex activity in shell construction. As observed by scanning microscopy, the shell consists of a single layer of sand grains which are organized into rosettes. The sand grains of the rosettes are different in size from those of flat areas between rosettes suggesting that the organism sorts these stones and places them according to size. Hydrofluoric acid treatment dissolves the sand but leaves a web of cement material intact. Examination of such acid treated specimens by transmission microscopy shows structure in the cement material of the shell, and granules of similar structure in the cell body. The rosette pattern observed differs from shell patterns in other species of Difflugia, and this suggests that shell structure may be species specific.     

Lower temperature and orthophosphate control of alkaline phosphatase in a cold-requiring plant

The elevated activity of alkaline phosphatase observed in roots and shoots of Verbascum rosettes grown in vitro at 15 °C may be the result of a reduced supply of orthophosphate. The rosette reveals greater activities of this enzyme under conditions of lower external phosphate or chilling temperature. Either of these treatments gives lower amounts of endogenous phosphate. The stimulation of phosphatase in tissues having lower levels of phosphate is thus a possible feature of other plant species which also display freezing tolerance.     

Tip cell growth and the frequency and distribution of particle rosettes in the plasmalemma: Experimental studies inFunaria protonema cells

Summary ComparingFunaria protonema tip cells of different age and of experimentally modified growth rate (by changing the light-dark-regime, by application of colchicine and of D₂O and by plasmolysis) we found that the site and intensity of growth are related closely to the distribution and frequency of particle rosettes in the PF of the plasma membrane. The results confirm previous suggestions that the rosettes are involved in cellulose fibril formation and that they have a rather short life time (about 10–15 minutes,Reiss et al. 1984). The appearance of rosettes seems to depend on the exocytosis of Golgi vesicle containing wall matrix material. Morphometric calculations suggest that each Golgi vesicle may incorporate one rosette into the plasmalemma in caulonema tip cells.     

A transient apical extracellular matrix relays cytoskeletal patterns to shape permanent acellular ridges on the surface of adult C. elegans

Epithelial cells secrete apical extracellular matrices to form protruding structures such as denticles, ridges, scales, or teeth. The mechanisms that shape these structures remain poorly understood. Here, we show how the actin cytoskeleton and a provisional matrix work together to sculpt acellular longitudinal alae ridges in the cuticle of adult C. elegans. Transient assembly of longitudinal actomyosin filaments in the underlying lateral epidermis accompanies deposition of the provisional matrix at the earliest stages of alae formation. Actin is required to pattern the provisional matrix into longitudinal bands that are initially offset from the pattern of longitudinal actin filaments. These bands appear ultrastructurally as alternating regions of adhesion and separation within laminated provisional matrix layers. The provisional matrix is required to establish these demarcated zones of adhesion and separation, which ultimately give rise to alae ridges and their intervening valleys, respectively. Provisional matrix proteins shape the alae ridges and valleys but are not present within the final structure. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion that shape a permanent and acellular matrix structure.     

An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations

Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates – allowing embryoid body formation – while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1⁺ dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2⁺ cortical neurons appearing after 1 week and upper-layer SATB2⁺ cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.KEY WORDS: Neocortical development, Dorsal forebrain model, hPSCs, Cocaine, Premature neuronal differentiation     

Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation

During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. Here, we show that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. We found that mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. We propose that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding.     

Myosin I is required for hypha formation in Candida albicans

The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5^S366D were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.     

Vannella primoblina n. sp. – an unusual species of the genus Vannella (Amoebozoa,Discosea, Vannellida) with pronounced dorsal ridges and folds

Amoebae of the order Vannellida (Amoebozoa, Discosea) have a fairly recognizable spatulate, fan-shaped or semi-circular outlines and a wide area of frontal hyaloplasm. They can be easily distinguished from the other groups of lobose amoebae even by light microscopy. The dorsal side of these amoebae is usually smooth and rarely bears ridges or folds, which are never numerous or regular. We have isolated an unusual species of vannellid amoebae, called Vannella primoblina n. sp. from a terrestrial substrate. It has well-developed dorsal relief consisting of regularly appearing folds and ridges. This amoeba superficially resembles members of the genus Thecamoeba. However, molecular analysis showed that this strain belongs to the genus Vannella. This finding indicates that dorsal folds may also be a characteristic of some species of vannellid amoebae and probably are a functional detail of the cell morphology rather than an apomorphy of Thecamoebida lineage. Overall outlines of the cell and the presence of the expanded frontal hyaline area remains the most reliable characters used to differentiate vannellid amoebae from other gymnamoebae lineages.     

Coelomocytes of earthworms: the T-cell-like rosette.

The coelomocytes forming spontaneous rosettes with sheep red blood cells (SRBC) were studied under various experimental conditions in lumbricus terrestris. The percentage of rosettes did vary with the pH, increased with the incubation period and remained constant at 4°C and at room temperature. The viability of the coelomocytes was a prerequisite for the formation of rosettes.The phenomenon was found to be stable, repeatable, and specific, thus suggesting rosettes are probably not an artefact.The coelomocytes forming rosettes were basophilic, nongranulated, and nonadhering cells with a large nucleus and little cytoplasm closely resembling vertebrate T-lymphocytes. The adhering cells did not form rosette.These results suggest that the coelomocytes forming rosettes could well be the evolutionary precursors or analogs of immunocyte rosettes forming cells of the vertebrates. Whether or not the coelomocyte receptors for the SRBC are similar to those of the T-cells of the vertebrate remains to be established.     

Zoonotic filariasis caused by Onchocerca dewittei japonica in a resident of Hiroshima Prefecture,Honshu, Japan

A female of Onchocerca sp. was found to be the probable causative agent of a subcutaneous nodule in the left knee of a 70-year-old man in a rural area of Hiroshima Prefecture, Honshu, the main island of Japan. We compared the characteristics of the agent with the features of the four previously suspected species found in cattle and horses in various parts of the world, as well as O. lupi and O. jakutensis that were suspected or proved, respectively, in zoonotic cases in Europe. In addition, the morphologic characteristics of this parasite were compared with those of the four Onchocerca species found in wild animals in Japan. Based on such characteristics as the large triangle ridges, the considerable distance between any two adjacent ridges, and the absence of inner cuticular striae in the longitudinal sections, we found the causative agent in the present case to be identical to the female of Onchocerca dewittei japonica. All five previous cases of zoonotic onchocerciasis in Japan had been found in Oita, Kyushu, the main southern island. This human case caused by O. dewittei japonica suggests that zoonotic onchocerciasis is liable to occur in rural areas in Japan where wild boar, Simulium vectors, and humans overlap.     

The type species of the ordovician trilobitesymphysurus: systematics,functional morphology and terrace ridges

The type of the widespread Ordovician trilobiteSymphysurus, S. palpebrosus Dalman, 1827, is redescribed from the type Swedish material; its distribution is documented.Symphysurus is one of very few trilobite genera to be found in both Ordovician Baltica and Gondwana, in more peripheral sites relative to the platform areas.S. palpebrosus has complex coaptative devices to ensure tight enrollment. Analysis of the functional morphology shows that it spent at least part of its life with thorax and pygidium buried in the sediment in the bumastoid stance, although it may have fed outside its burrow. Terrace ridges are well developed on certain parts of the cuticle; their positioning and geometry shows that only a few of them could have functioned in direct engagement with the sediment. Those associated with petaloid thoracic facets may have permitted respiration in the enrolled condition.