The role of Wnt signalling in the development of somites and neural crest

The Wnt family of secreted signalling molecules controls a wide range of developmental processes in all metazoans. In this investigation we concentrate on the role that members of this family play during the development of (1) the somites and (2) the neural crest. (3) We also isolate a novel component of the Wnt signalling pathway called Naked cuticle and investigate the role that this protein may play in both of the previously mentioned developmental processes. (1) In higher vertebrates the paraxial mesoderm undergoes a mesenchymal-to-epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists which result in the segmental plate being unable to form somites. These results show that Wnt6, the only member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. (2) The neural crest is a population of multipotent progenitor cells that arise from the neural ectoderm in all vertebrate embryos and form a multitude of derivatives including the peripheral sensory neurons, the enteric nervous system, Schwann cells, pigment cells and parts of the craniofacial skeleton. The induction of the neural crest relies on an ectodermally derived signal, but the identity of the molecule performing this role in amniotes is not known. Here we show that Wnt6, a protein expressed in the ectoderm, induces neural crest production. (3) The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement (Pandur et al. 2002b). Recent work has identified the protein Naked cuticle as an intracellular switch promoting the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked cuticle-1 (cNkd1) and demonstrate that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly our study shows that the expression of cNkd1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum

Ascaris suum is one of the most prevalent nematode parasites in pigs and causes significant economic losses, and also serves as a good model for A. lumbricoides, the large roundworm of humans that is ubiquitous in developing countries and causes malnutrition, stunted growth and compromises immunity to other pathogens. New treatment options for Ascaris infections are urgently needed, to reduce reliance on the limited number of synthetic anthelmintic drugs. In areas where Ascaris infections are common, ethno-pharmacological practices such as treatment with natural plant extracts are still widely employed. However, scientific validation of these practices and identification of the active compounds are lacking, although observed effects are often ascribed to plant secondary metabolites such as tannins. Here, we extracted, purified and characterised a wide range of condensed tannins from diverse plant sources and investigated anthelmintic effects against A. suum in vitro. We show that condensed tannins can have potent, direct anthelmintic effects against A. suum, as evidenced by reduced migratory ability of newly hatched third-stage larvae and reduced motility and survival of fourth-stage larvae recovered from pigs. Transmission electron microscopy showed that CT caused significant damage to the cuticle and digestive tissues of the larvae. Furthermore, we provide evidence that the strength of the anthelmintic effect is related to the polymer size of the tannin molecule. Moreover, the identity of the monomeric structural units of tannin polymers may also have an influence as gallocatechin and epigallocatechin monomers exerted significant anthelmintic activity whereas catechin and epicatechin monomers did not. Therefore, our results clearly document direct anthelmintic effects of condensed tannins against Ascaris and encourage further in vivo investigation to determine optimal strategies for the use of these plant compounds for the prevention and/or treatment of ascariosis.

Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum

Background: Plant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life-cycles stages of O. dentatum. Methods: Extracts and purified fractions were prepared from five plants containing CT and analysed by HPLC-MS. Anthelmintic activity was assessed at five different stages of the O. dentatum life cycle; the development of eggs to infective third-stage larvae (L3), the parasitic L3 stage, the moult from L3 to fourth-stage larvae (L4), the L4 stage and the adult stage. Results: Free-living larvae of O. dentatum were highly susceptible to all five plant extracts. In contrast, only two of the five extracts had activity against L3, as evidenced by migration inhibition assays, whilst three of the five extracts inhibited the moulting of L3 to L4. All five extracts reduced the motility of L4, and the motility of adult worms exposed to a CT-rich extract derived from hazelnut skins was strongly inhibited, with electron microscopy demonstrating direct damage to the worm cuticle and hypodermis. Purified CT fractions retained anthelmintic activity, and depletion of CT from extracts by pre-incubation in polyvinylpolypyrrolidone removed anthelmintic effects, strongly suggesting CT as the active molecules. Conclusions: These results suggest that CT may have promise as an alternative parasite control option for O. dentatum in pigs, particularly against adult stages. Moreover, our results demonstrate a varied susceptibility of different life-cycle stages of the same parasite to CT, which may offer an insight into the anthelmintic mechanisms of these commonly found plant compounds.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

Morphometrical, biochemical and molecular tools for assessing biodiversity. An example in Plebeia remota (Holmberg, 1903) (Apidae, Meliponini)

We see today many efforts to quantify biodiversity in different biomes. It is very important then to develop and to apply other methodologies that allow us to assess biodiversity. Here we present an example of application of three tools with this goal. We analyzed two populations of Plebeia remota from two distinct biomes that already showed several differences in morphology and behavior. Based on these differences, it has been suggested that the populations of Cunha and Prudentopolis do not represent a single species. In order to verify the existence or absence of gene flow between these two groups, we characterized the patterns of mtDNA through RFLP, the patterns of wing venation through geometric morphometry, and the cuticular hydrocarbons through gas chromatography-mass spectrometry. We used bees collected in these two locations and also from colonies which have being kept for around 9 years at Sao Paulo University. We found six different haplotypes in these specimens, of which three of them occurred exclusively in the population of Cunha and three only in the Prudentopolis population. The fact that the populations do not share haplotypes suggests no maternal gene flow between them. The two populations were differentiated by the pattern of the wing veins. They also had different mixtures of cuticle hydrocarbons. Furthermore it was shown that the colonies kept at the university did not hybridize. These two groups may constitute different species. We also show here the importance of using other methodologies than traditional taxonomy to assess and understand biodiversity, especially in bees.

Embryonic Development of Ampheres leucopheus and lporangaia pustulosa (Arachnida: Opiliones: Gonyleptidae)

The first studies concerning the embryonic development of harvestmen started in the late 19th century, and focused mostly on holarctic species, and only three species of the suborder Laniatores (the largest, among the four suborders considered presently) were studied. Moreover, the last studies on embryology of harvestmen were made during the late 1970s. This study focused on the embryonic development of Ampheres leucopheus (Gonyleptidae, Caelopyginae) and Iporangaia pustulosa (Gonyleptidae, Progonyleptoidellinae). The embryonic development was followed in the field, by taking daily photographs of different eggs during about 2 months. When laid, eggs of A. leucopheus and I pustulosa have approximately 1.13 and 1.30 mm in diameter, respectively, and the second is embedded in a large amount of mucus. The eggs grow, mainly due to water absorption at the beginning of the process, and they reach a diameter of about 1.35 and 1.59 mm, respectively, close to hatching. It took, respectively, 29-56 days and 35-66 days from egg laying to hatching. For the description of the embryonic development, we use photographs from the field, SEM micrographs, and histological analysis. This allowed us, for instance, to document the progression of structures and pigmentation directly from live embryos in the field, and to record microstructures, such as the presence of perforations in the cuticle of the embryo in the place where eyes are developing. Yet, contrary to what was expected in the literature, we record an egg tooth in one of the studied laniatoreans. J. Exp. Zool. (Mol. Dev. Evol) 314B:489-502, 2010. (C) 2010 Wiley-Liss, Inc.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood-induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood-feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin-binding domains with 6 conserved Cys and may also present mucin-like domains (regions expected to be highly O-glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin-like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood-feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

Ultramorphology and histochemistry of fat body cells from last Instar larval of the Pachycondyla (=Neoponera) villosa (Fabricius) (Formicidae: Ponerinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morfologia de frutos de goiabeira

A eficiência na absorção de nutrientes pelos frutos pode sofrer interferências em função da estrutura morfológica presente. Assim, frente à importância do conhecimento da morfologia dos frutos para um manejo mais adequado dos pomares em termos de adubação foliar, ou de tratamentos pós-colheita para ampliar a vida útil dos mesmos, desenvolveu-se o presente estudo, cujo objetivo foi descrever a morfologia do pericarpo dos frutos de goiabeira. Para isto, amostras do pericarpo de goiabas (cv. Paluma) foram observadas ao microscópio ótico e eletrônico de varredura. Algumas das características observadas, como a presença de cutícula espessa, cêra epicuticular, três camadas subepidérmicas de células compactas e grande quantidade de esclereídeos, bem como a presença esporádica e dispersa de estômatos, podem constituir-se em barreiras para a absorção e movimentação de nutrientes e substâncias aplicadas aos frutos de goiabeira.

An evergreen neotropical savanna tree (Gochnatia polymorpha, Asteraceae) produces different dry- and wet-season leaf types

Gochnatia polymorpha (Less.) Cabrera is a widespread tree species found in different physiognomies of neotropical savanna (cerrado) formations of south-eastern Brazil. The present study describes some leaf anatomical characteristics of this species as a function of the time of leaf flush, during dry or wet seasons. This species presents anatomical plasticity in the cuticle, palisade parenchyma and abaxial epidermis as well as in stomatal size and stomatal and trichome density, which are leaf structures linked with water-status control. Leaf structure changed to suit the particular environmental conditions during dry and wet seasons. The production of different wet-and dry-season leaf types in G. polymorpha could be a response to drought and an adaptation to environmental constraints in the cerrado.

Aerial stem and leaf morphoanatomy of some species of Smilax

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Usos potenciais de moringa oleifera lam., uma matriz para produção de biodiesel e tratamento de água no semiárido nordestino

The current environmental crisis demands transformations in the relations among society, nature and development, considering sustainability. In this context, an important theme is replacing fossil fuels with biofuels, such as biodiesel. Moringa oleifera Lam. is a species that can be used as a raw material to produce biodiesel. Besides, it is a multiple purposes plant, which can be used also in water treatment. Thus, the aims of this work were to analyze the anatomical adaptations found in the stem and in the leaf and the seed s oil stores of M. oleifera., to investigate chemical characteristics of M. oleifera s seed oil, considering biodiesel production, and to evaluate the coagulation activity of these seeds in water treatment. Semipermanent histological laminas were made and it follows that the stem has thick cuticle, stomata whose cells guard are below the epidermis line, hollow medulla, druses and tector trichomes as adaptations to climate and soil conditions in which the species is found and the leaf is dorsiventral and it has thick cuticle, tector trichomes and druses. The seed has great reserves of oil. These features favor the use of Moringa oleifera Lam. as a raw material to produce biodiesel in Brazil s Northeast semiarid region. Chemical analysis were made through oil solvent extraction using mechanic stirrer. The oil was analyzed in UV spectrophotometer. A transesterification was made and biodiesel was analyzed in gas chromatography. Oil yield was high and good quality biodiesel was obtained. To evaluate seeds coagulantion activity, coagulation and flocculation essays in jartest were made, using seed extract to treat raw water. Seeds were efficient in cogulation process to treat water. So, they can be used in rudimentary systems or as a raw material to coagulant proteins extraction, as an alternative to traditional coagulants. M. oleifera has characteristics that favor its use to biodiesel production and water treatment

Ontogenesis, structure and ultrastructure of Hymenaea stigonocarpa (Fabaceae : Caesalpinioideae) colleters

The genus Hymenaea is characterized by a great diversity of secretory structures, but there are no reports of colleters yet. The objectives of this study are to report the occurrence and describe the origin and structure of colleters in Hymenaea stigonocarpa Mart. ex Hayne. Shoot apex samples were collected, fixed, and processed for light microscopy, scanning electron microscopy, and transmission electron microscopy as per usual methods. Colleters occur predominantly on the stipule's adaxial side. These structures are found at the base on a narrow strip, corresponding to the median vein up to half the length of the stipule. When present on the abaxial side, they are concentrated at the base and restricted to the margins. Colleters develop from the protoderm; they are elongate and club-shaped. Their body has no stratification; their surface cells differ from the inner cells only in position and presence of cuticle. Colleter cells have thin walls, dense cytoplasm, large nuclei, many mitochondria, rough endoplasmic reticulum, and abundant dictyosomes. Histochemical tests with Ruthenium red showed pectic compounds in the cytosol. In H. stigonocarpa, colleter arrangement is compatible with the hypothesis that they protect shoot apex. In this species, protection is reinforced by the sheath formed by the stipule pairs.