Muscle formation in the sexual genetation of Intoshia variabili (Orthonectida)

Muscle formation in Intoshia variabili (Orthonectida) has been studied in the course of development of the sexual (free-living) specimen. The muscle system originates in the early embryogenesis as a distinct continuous layer located between the outer cell layer and the inner cell mass. Later this cell layer disintegrates into separate muscle strips. The presence of a distinct muscle system in Orthonectida and the pattern of its development evidences for placing this group into Triploblastica rather than into Diploblastica.     

Fine structure and development of the cuticle of Intoshia variabili (Orthonectida)

The body of free-swiming mature Intoshia variabili (Orthonectida) is covered by a thin cuticle, 0.3 μm thick. The cuticle is formed at the time when the orthonectid embryos develop in the plasmodium. The process of cuticle formation begins just after the first cilia begin to appear at the surface of the ciliated cells. At first, small extensions of the cell membrane appear at the surface of the cell, more or less parallel to the cell surface. As they develop futher, they stand up, and amorphic material begins to appear between them. The extensions then become microvilli and obtain their final shape, with a small subdistal swelling and a narrower distal part. They are situated very regularly on the surface of the cell. After the microvilli have obtained their final form, material between them begins to get its final structure typical of the adult form. During the period when the mature orthonectid begins to leave the plasmodium and emerge from the host, the regular microvilli begin to disappear, and only small irregular extensions are present under the cuticle on the surface of the cell. During the process of cuticle formation a large amount of smooth endoplasmic reticulum develops in the cells, but once the cuticle is formed it gradually disappears.     

Nuclei in the plasmodium of Intoshia variabili (Orthonectida) as revealed by DAPI staining

DAPI staining of wholeamounts was used to reveal the parasitic plasmodium of the orthonectid Intoshia variabili in its host, the turbellarian Macrorhynchus crocea. The nuclei of the parasite differ drastically from those of the host in size, morphology, and the estimated DNA content. Our findings indirectly support the idea that the orthonectid plasmodium is a distinct parasitic organism, rather than modified host cells.     

Fine Structure of the Female Intoshia variabili(Alexandrov & Sljusarev) (Mesozoa: Orthonectida)

The morphology and fine structure of female Intoshia variabili, new combination for Rhopalura variabiliAlexandrov & Sljusarev, 1992, were studied with transmission electron microscopy. The body surface is covered with a 3-layered cuticula, under which is a layer of ciliated + non-ciliated cells arranged in alternating rings around the body. Ciliated cells have lateral extensions that intercalate with the non-ciliated cells. The kinetosome of each cilium has two longitudinally oriented cross-striated rootlets. The outer surface of the ciliated cells is covered with small tubercles, and the cytoplasm of these cells contains granules, vacuoles, mitochondria, fibrillar structures and lamellary bodies. A band of dense fibrils passes through the upper part of each ring of cells, going from one cell junction to another, encircling the entire body. Between the layer of ciliated + non–ciliated cells and the oocytes, elongated contractile cells from 4–5 longitudinal columns and 1 ring, the latter at the level of ciliated rings 7–9. The contractile cells contain thick and thin longitudinally oriented fibrils. The oocytes contain a large nucleus, numerous mitochondria, electron–dense granules and 1–2 spherical structures. An anteriorly situated, ciliated goblet–like receptor, not described for any other orthonectids, consists of three closely apposed cells, the upper part of which contains densely packed cilia. The genital pore opens through a non–ciliated cell and is surrounded by several cells with granules.     

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.     

Cladistic biogeography of the Mexican transition zone

Biogeographic relationships among nine montane areas of endemism across the transition zone between North and South America are analysed cladistically based on phylogenetic hypotheses of thirty‐three resident monophyletic taxa of insects, fish, reptiles, and plants. Areas of endemism include the Arizona mountains (AZ), Sonoran Desert (SD), Sierra Madre Occidental (OCC), southern Sierra Madre Occidental (SOC), Sierra Madre Oriental (ORI), Sierra Transvolcanica (TRAN), Sierra Madre del Sur (SUR), Chiapan‐Guatemalan Highlands (CGH), and Talamancan Cordillera (TC). Area relationships are summarized using Brooks Parsimony Analysis and Assumption 0, with the former resulting in more defensible biogeographic hypotheses. Areas of endemism are dividable into two monophyletic groups; a northern group including AZ, SD, OCC, and ORI, and a southern group consisting of TC, CGH, TRAN, SUR, and the isolated southern regions of the Sierra Madre Occidental (SOC). The northern set of areas are characterized by recent, probably Pleistocene, isolation and prevalent widespread species, whereas the southerly areas probably diverged after Pliocene closure of the Panamanian isthmus. The southern areas are redundantly represented on many of the taxon‐area cladograms by endemic species, indicative of much higher levels of endemism in the Sierra Transvolcanica and further south. Use of a general area cladogram in such a transition zone permits explicit exploration of biogeographic patterns and establishes a predictive framework for taxonomy and conservation prioritization.     

Analysis of possible mechanisms of orthonectid emergence from their hosts

In the present study authors claim that the adult orthonectids can not move through host tissues by themselves. In various species of these enigmatic parasites there are at least two different mechanisms of emission of males and females from the host body. Intoshia linei, the orthonectid from Lineus ruber (Heteronemertini), and Intoshia variabili, the parasite of a flatworm Macrorhynchus crocea, realize the first way of emission. The plasmodium of these species forms tube-like outgrowths, which pierce the host tissues reaching the host body surface. The cytoplasm structure of these outgrowths differs from the cytoplasm of the central mass of plasmodium. Small mitochondria with electron dense matrix, lipid granules and vesicular bodies being common in the central part are absent in these outgrowths. Plasmodial outgrowths reach the host body surface and adult orthonectids move inside them using their cilia and stopping from time to time. The plasmodial outgrowths penetrate the ciliated epithelium, then males and females leave the host. Duration of emission may vary in different species from 6 to 13 days. The second mechanisms of emission is common for the orthonectid parasites of mollusks. Our observations of Rhopalura philinae from the gastropod Philine scabra lead to the conclusion that males and females leave their host practically simultaneously. When the plasmodium attains the terminal stage of its development most of the host entrails are already displaced by plasmodial mass. It causes breaks in host body walls and hence to emission of sexual individuals. During this process, which lasts about 24 hours, the mollusk dies. The same mechanism was observed in Rhopalura littoralis--parasite of the gastropod Onoba aculeus. Our investigations of emission ways reveal that the plasmodium of orthonectids has a potency of directing growth and can form certain structures. The process of forming the plasmodial outgrowths is coordinated in time and space. These outgrowths have certain directions inside the host body and the maturation of sexual individuals is clear related with the development of plasmodium outgrowth system. Our results suggest that forming of plasmodial outgrowths is an element of development of the united and highly integrated system. It is necessary to emphasize the capability of plasmodium to accomplish such morphogenetic transformations. This fact argues that plasmodium is a part of parasite organism and not host cells modified, like some experts supposed.     

Ciliary disorder of the skeleton

In the last 10 years, the primary cilia machinery has been implicated in more than a dozen disorders united as ciliopathies, including skeletal dysplasias, such as Jeune syndrome and short rib-polydactyly type III. Indeed, primary cilia play a vital role in transduction of signals in the hedgehog pathway that is especially important in skeletal development. In this review, we focus on skeletal conditions belonging to the ciliopathy group: the short rib-polydactyly group (SRPs) that includes Verma-Naumoff syndrome (SRP type III), Majewski syndrome (SRP type II), Jeune syndrome (ATD), as well as Ellis-van Creveld syndrome (EVC), the Sensenbrenner syndrome, and, finally, Weyers acrofacial dysostosis. Today, 10 different genes have been identified as responsible for seven "skeletal" ciliopathies. Mutations have been identified in dynein motor (DYNC2H1), in intraflagellar transport (IFT) complexes (IFT80, IFT122, IFT43, WDR35, WDR19, and TTC21B) as well as in genes responsible for the basal body (NEK1, EVC, and EVC2). The wide clinical variability observed for an individual ciliopathy gene supports the development of exome strategy specifically dedicated to cilia genes to identify mutations in this particularly heterogeneous group of disorders.     

Distribution of microaerophilic bacteria through the oxic-anoxic transition zone of lagoon sediments

In marine sediments, where soluble gases diffuse only very slightly, many organisms struggle for molecular oxygen. Microaerophilic bacteria, able to grow at reduced pO₂ between 0.2 and 12%, have an advantage. Distribution of aerobes, microaerophiles and anaerobes was compared with the oxygen gradient in seawater and sediment samples collected in a northern Mediterranean lagoon. In the near bottom seawater and in the 0–10 mm upperlayer of sediment, the microaerophilic counts were less than 1% of aerobe densities. In the 10–15 mm zone, these two groups were equivalent in density (1 × 10⁵ cells ml^–1). As expected, the microaerophiles took advantage of their low oxygen tension requirements in the subsurface sediments, between the well aerated zone (0–5 mm depth) and the low redox potential zone. Then, beyond a depth of 20 mm, the anaerobes prevailed in this sandy clay.     

气候变暖与荒漠-草原过渡带植物动态响应灰色分析--以美国新墨西哥州Sevilleta生态过渡带为例

在当前全球气候变暖的形势下,地表生态系统表现出不同的响应。干旱-半干旱地区的生态过渡带的生物反应更为敏感。美国新墨西哥州中部的荒漠-草原生态过渡带是监测全球气候变化和人类活动对生物影响的重要区域之一。本文以Sevilleta荒漠．草原生态过渡带为例,运用灰色系统分析方法．研究在当前全球气候变暖的条件下,1989～1998年10年间该生态过渡带的两种共存的植物优势种(Bouteloua eripoda)和(Bouteloua grncilis)历年的种群密度、组合形式和变化趋势,分析引起这些变化的主要气候因子。结果表明,Sevilleta生态过渡带中来自荒漠草原的优势物种Bouteloua eripoda的密度有上升的趋势,来自大草原的优势物种Bouteloua gracilis的密度有下降的趋势,它们的密度比大于1且有上升的趋势;在影响种群密度变化的降水、最高温度、最低温度、湿度的4个气候因子中,温度因子起着重要的作用。由此可以认为,随着气候的变暖,本区来自荒漠地区的优势植物种在荒漠-草原群落过渡带中将逐渐处于强优势地位,生态过渡带将有可能被荒漠草原所替代。     

Plant communities in the terrestrial-aquatic transition zone in the paramo of Chingaza, Colombia

Plant communities in the terrestrial-aquatic transition zone in the paramo of Chingaza, Colombia. High Andean paramo ecosystems are an important water resource for many towns, and major cities in this region. The aquatic and wetland vegetation of different paramo lakes, pond, swamps and bogs was studied according to the classical phytosociological approach, which is based on homogenous stands, but excludes any border phenomena or transitional zone. The present research aimed at determining the aquatic and wetland vegetation along different moisture gradients. A total of 89 species in 30 transects were reported, of which Crassula venezuelensis, Carex honplandii, Callitriche nubigena, Eleocharis macrostachya, Ranunculus flagelliformis, R. nubigenus, Eleocharis stenocarpa, Galium ascendens y Alopecurus aequalis were present in more than one third of the transects. Numerical classification and indicator species analysis resulted in the definition of the next 18 communities: 1) Calamagrostis effusa, 2) Sphagnum cuspidatum, 3) Cyperus rufus, 4) Eleocharis stenocarpa, 5) Carex acutata, 6) Poa annua,7) Valeriana sp., 8) Ranunculus flagelliformis, 9) Carex bonplandii, 10) Festuca andicola. 11) Muhlenbergia fustigiata, 12) Elatine paramoana, 13) Iso?tes palmeri, 14) Crassula venezuelensis, 15) Lilaeopsis macloviana, 16) Callitriche nubigena, 17) Potamogeton paramoanus and 18) Potamogeton illinoensis. The ordination of communities reveals the presence of three different aquatic-terrestrial gradients which are related to the life form structure of species that characterized the various communities. We concluded that patchiness and heterogeneity of the vegetation is mainly the result of alterations caused by human activities (burning, cattle raise and material extraction for road and dam construction).     

The structure and mechanical properties of the mitral valve leaflet-strut chordae transition zone

Biaxial testing, histological measurements and theoretical continuum mechanics modeling were employed to investigate the structure and mechanical properties of the mitral valve leaflet-strut chordae transition zone (LCT). The results showed that geometry changes and collagen fiber angle distribution contribute to variations in mechanical properties in the LCT zone. A simple three-coefficient exponential constitutive law was able to simulate the variation in stress-stretch behavior in the LCT zone by spatially varying a single coefficient and incorporating collagen fiber angle and degree of alignment. This quantitative information can greatly improve the predictions from biomechanical valve models by incorporating regional variations of structure and properties in the mitral leaflet-chordae tendineae system. These data provide the foundation for a computational model for studying stress distributions before and following chordal rupture, which may indicate the underlying reasons for the development of valve insufficiency in patients.     

The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule-based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal-most region of the axoneme, termed the transition zone, which is characterized by Y-shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.     

Ciliary adaptations for the propulsion of mucus

The cilia that propel mucus are specialised for the function in their arrangement, length, some details of structure, beat pattern, beat cycle characteristics, metachronal coordination, local control of beat rate by response to mechanical stimulation and generalised control of beat rate by neurohormones. These features are matched to the properties of the visco-elastic mucus gel that is propelled at the ciliary tips above a low-viscosity periciliary layer whose depth must be regulated within defined limits.     

Morphological Aspects of Ciliary Motility

In Elliptio complanatus lateral cilia, two distinct patterns of filament termination can be discerned. In one case, all nine filaments are present and all are single; in the second, at least one filament is missing but doublets are still present. These probably represent different configurations within one cilium in different stroke positions; to get from one to the other, some peripheral filaments must move with respect to others. The data are consistent with the hypothesis that the filaments themselves do not change length, but rather slide past one another to accommodate increasing curvature. The bent regions of the cilium are in the form of circular arcs. In a few cases, apparent displacement of filaments at the tip (Δl) can be shown to be accounted for if we assume that all differences are generated within these arcs. The displacement per degree of bend is 35 A. Regions of bent arc are initially confined to the base of the cilium but move up the shaft as straight regions appear below them. From the relationship between arc length and radius of curvature, a shaft length that is the unit that initially bends and slides may be defined. Quantal displacements of the length of one 14S dynein may perhaps occur at sites between filaments at opposite sides of such a unit as sliding occurs.     

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.     

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.     

Bioinformatic analysis of ciliary transition zone proteins reveals insights into the evolution of ciliopathy networks

Background

Cilia are critical for diverse functions, from motility to signal transduction, and ciliary dysfunction causes inherited diseases termed ciliopathies. Several ciliopathy proteins influence developmental signalling and aberrant signalling explains many ciliopathy phenotypes. Ciliary compartmentalisation is essential for function, and the transition zone (TZ), found at the proximal end of the cilium, has recently emerged as a key player in regulating this process. Ciliary compartmentalisation is linked to two protein complexes, the MKS and NPHP complexes, at the TZ that consist largely of ciliopathy proteins, leading to the hypothesis that ciliopathy proteins affect signalling by regulating ciliary content. However, there is no consensus on complex composition, formation, or the contribution of each component.

Results

Using bioinformatics, we examined the evolutionary patterns of TZ complex proteins across the extant eukaryotic supergroups, in both ciliated and non-ciliated organisms. We show that TZ complex proteins are restricted to the proteomes of ciliated organisms and identify a core conserved group (TMEM67, CC2D2A, B9D1, B9D2, AHI1 and a single TCTN, plus perhaps MKS1) which are present in >50% of all ciliate/flagellate organisms analysed in each supergroup. The smaller NPHP complex apparently evolved later than the larger MKS complex; this result may explain why RPGRIP1L, which forms the linker between the two complexes, is not one of the core conserved proteins. We also uncovered a striking correlation between lack of TZ proteins in non-seed land plants and loss of TZ-specific ciliary Y-links that link microtubule doublets to the membrane, consistent with the interpretation that these proteins are structural components of Y-links, or regulators of their formation.

Conclusions

This bioinformatic analysis represents the first systematic analysis of the cohort of TZ complex proteins across eukaryotic evolution. Given the near-ubiquity of only 6 proteins across ciliated eukaryotes, we propose that the MKS complex represents a dynamic complex built around these 6 proteins and implicated in Y-link formation and ciliary permeability.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-531) contains supplementary material, which is available to authorized users.     

A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia.?The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.