Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest

In pre-B lymphocytes, productive rearrangement of Ig light chain genes allows assembly of the B cell receptor (BCR), which selectively promotes further developmental maturation through poorly defined transmembrane signaling events. Using a novel in vitro system to study immune tolerance during development, we find that BCR reactivity to auto-antigen blocks this positive selection, preventing down-regulation of light chain gene recombination and promoting secondary light chain gene rearrangements that often alter BCR specificity, a process called receptor editing. Under these experimental conditions, self-antigen induces secondary light chain gene rearrangements in at least two-thirds of autoreactive immature B cells, but fails to accelerate cell death at this stage. These data suggest that in these cells the mechanism of immune tolerance is receptor selection rather than clonal selection.

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

Sexing of murine and bovine embryos by developmental high-titer arrest induced by H-Y antisera

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12 h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos. (c) 2004 Elsevier B.V. All rights reserved.

Ins-4 and daf-28 function redundantly to regulate C. elegans L1 arrest

© 2014 The Authors.Caenorhabditis elegans larvae reversibly arrest development in the first larval stage in response to starvation (L1 arrest or L1 diapause). Insulin-like signaling is a critical regulator of L1 arrest. However, the C. elegans genome encodes 40 insulin-like peptides, and it is unknown which peptides participate in nutritional control of L1 development. Work in other contexts has revealed that insulin-like genes can promote development ("agonists") or developmental arrest ("antagonists"), suggesting that such agonists promote L1 development in response to feeding. We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified thirteen candidate agonists and eight candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (. daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists is largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in control of L1 development. Transcriptional regulation of candidate agonists is most significant in the intestine, as if internal nutrient status is a more important influence on transcription than sensory perception. Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 developmental dynamics, though simultaneous disruption of ins-4 and daf-28 increases survival of L1 arrest. Furthermore, overexpression of ins-4, ins-6 or daf-28 alone decreases survival and promotes cell division during starvation. These results suggest extensive functional overlap among insulin-like genes in nutritional control of L1 development while highlighting the role of ins-4, daf-28 and to a lesser extent ins-6.

To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest.

It is widely appreciated that larvae of the nematode Caenorhabditis elegans arrest development by forming dauer larvae in response to multiple unfavorable environmental conditions. C. elegans larvae can also reversibly arrest development earlier, during the first larval stage (L1), in response to starvation. "L1 arrest" (also known as "L1 diapause") occurs without morphological modification but is accompanied by increased stress resistance. Caloric restriction and periodic fasting can extend adult lifespan, and developmental models are critical to understanding how the animal is buffered from fluctuations in nutrient availability, impacting lifespan. L1 arrest provides an opportunity to study nutritional control of development. Given its relevance to aging, diabetes, obesity and cancer, interest in L1 arrest is increasing, and signaling pathways and gene regulatory mechanisms controlling arrest and recovery have been characterized. Insulin-like signaling is a critical regulator, and it is modified by and acts through microRNAs. DAF-18/PTEN, AMP-activated kinase and fatty acid biosynthesis are also involved. The nervous system, epidermis, and intestine contribute systemically to regulation of arrest, but cell-autonomous signaling likely contributes to regulation in the germline. A relatively small number of genes affecting starvation survival during L1 arrest are known, and many of them also affect adult lifespan, reflecting a common genetic basis ripe for exploration. mRNA expression is well characterized during arrest, recovery, and normal L1 development, providing a metazoan model for nutritional control of gene expression. In particular, post-recruitment regulation of RNA polymerase II is under nutritional control, potentially contributing to a rapid and coordinated response to feeding. The phenomenology of L1 arrest will be reviewed, as well as regulation of developmental arrest and starvation survival by various signaling pathways and gene regulatory mechanisms.

From cell death to embryo arrest: Mathematical models of human preimplantation embryo development

Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro. The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos. We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this. This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages.

Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

Falha na sexagem por inibição do desenvolvimento de embriões bovinos produzidos in vitro com anticorpos anti H-Y

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle

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

Interação núcleo-citoplasmática em embriões e expressão de genes imprinted em fetos bovinos produzidos in vivo, in vitro e partenogenéticos

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

Untersuchung neuer hochwirksamer neuroprotektiver Antioxidantien in experimentellen Modellen der Parkinsonschen Krankheit

Bei der Parkinsonschen Krankheit kommt es zu einer selektiven Degeneration der dopaminergen Neurone in der Substantia nigra pars compacta. Die Rolle des oxidativen Stresses in der Pathogenese dieser Erkrankung konnte an post mortem Untersuchungen der Parkinson-Patienten, wie auch an zahlreichen in vitro und in vivo Modellen bestätigt werden. Die Anwendung von Antioxidantien wurde als therapeutische Strategie der Parkinsonschen Krankheit vorgeschlagen. In dieser Hinsicht wurden bereits antioxidative Substanzen in klinischen Studien evaluiert. Klinische Studien mit Antioxidantien haben jedoch bislang nur wenig überzeugende Ergebnisse erbracht, mit Ausnahme des Einsatzes des Ubichinons (Coenzym Q). Eine kritische Analyse der klinischen Studien lässt zusammenfassen, dass auf Seiten der verwendeten Antioxidantien noch massiver Optimierungsbedarf besteht. Für einen erfolgreichen therapeutischen Einsatz von Antioxidantien bei dieser Krankheit sind folgende Eigenschaften der Substanzen von höchster Bedeutung: i) maximale neuroprotektive Aktivität bei geringen Dosen; ii) geringe Nebenwirkungen; iii) eine hohe Blut-Hirn-Schrankengängigkeit.In dieser Arbeit wurde das neuroprotektive Potential von drei Bisarylimin-basierten antioxidativen Strukturen (Phenothiazin, Iminostilben und Phenoxazin) in in vitro und in vivo Parkinson-Modellsystemen evaluiert. Beide experimentellen Modelle basieren auf der Wirkung der mitochondrialen Komplex I Inhibitoren 1-Methyl-4-Phenylpyridin (MPP+) und Rotenon, welche pathophysiologische Charakteristika der Parkinsonschen Krankheit reproduzieren. Unsere in vitro Untersuchungen an primären Neuronen des Mittelhirns und der klonalen SH-SY5Y-Neuroblastomazelllinie konnten zeigen, dass die Komplex I Inhibition krankheitsspezifische zelluläre Merkmale induziert, wie die Abnahme der antioxidativen Verteidigungskapazität und Verlust des mitochondrialen Membranpotentials. Zusätzlich kommt es in primären Neuronen des Mittelhirns zur selektiven Degeneration dopaminerger Neurone, welche in der Parkinsonschen Erkrankung besonders betroffen sind. Ko-Inkubation der in vitro Modelle mit Phenothiazin, Iminostilben und Phenoxazin in niedrigen Konzentrationen (50 nM) halten die pathologischen Prozesse fast vollständig auf. In vivo Untersuchungen am MPP+- und Rotenon-basierten Caenorhabditis elegans (C. elegans) Modell bestätigen das neuroprotektive Potential der Bisarylimine. Hierfür wurde eine transgene C. elegans Linie mithilfe einer dopaminerg spezifischen DsRed2- (Variante des rot fluoreszierenden Proteins von Discosoma sp.)-Expression und pan-neuronaler CFP- (cyan fluoreszierendes Protein)-Expression zur Visualisierung der dopaminergen Neuronenpopulation in Kontrast zum Gesamtnervensystem erstellt. Behandlung des C. elegans mit MPP+ und Rotenon im larvalen und adulten Stadium führt zu einer selektiven Degeneration dopaminerger Neurone, sowie zum Entwicklungsarrest der larvalen Population. Die dopaminerge Neurodegeneration, wie auch weitere phänotypische Merkmale des C. elegans Modells, können durch Phenothiazin, Iminostilben und Phenoxazin in niedrigen Konzentrationen (500 nM) komplett verhindert werden. Ein systemischer Vergleich aromatischer Bisarylimine mit bekannten, gut charakterisierten Antioxidantien, wie α-Tocopherol (Vitamin E), Epigallocatechingallat und β-Catechin, zeigt, dass effektive Konzentrationen für Phenothiazin, Iminostilben und Phenoxazin um Zehnerpotenzen niedriger liegen im Vergleich zu natürlichen Antioxidantien. Der Wirkungsmechanismus der Bisarylimine konnte in biochemischen und in vitro Analysen, sowie in Verhaltensuntersuchungen an C. elegans von der Wirkungsweise strukturell ähnlicher, neuroleptisch wirkender Phenothiazin-Derivate differenziert werden. Die Analyse des dopaminerg-gesteuerten Verhaltens (Beweglichkeit) in C. elegans konnte verdeutlichen, dass antioxidative und Dopaminrezeptor-bindende Eigenschaften der Bisaryliminstrukturen sich gegenseitig ausschließen. Diese qualitativen Merkmale unterscheiden Bisarylimine fundamental von klinisch angewandten Neuroleptika (Phenothiazin-Derivate), welche als Dopaminrezeptor-Antagonisten zur Behandlung psychischer Erkrankungen klinisch eingesetzt werden.Aromatische Bisarylimine (Phenothiazin, Iminostilben und Phenoxazin) besitzen günstige strukturelle Eigenschaften zur antioxidativ-basierter Neuroprotektion. Durch die Anwesenheit der antioxidativ wirkenden, nicht-substituierten Iminogruppe unterscheiden sich Bisarylimine grundlegend von neuroleptisch-wirkenden Phenothiazin-Derivaten. Wichtige strukturelle Voraussetzungen eines erfolgreichen antioxidativen Neuropharmakons, wie eine hohe Radikalisierbarkeit, die stabile Radikalform und der lipophile Charakter des aromatischen Ringsystems, werden in der Bisaryliminstruktur erfüllt. Antioxidative Bisarylimine könnten in der Therapie der Parkinsonschen Krankheit als eine effektive neuroprotektiv-therapeutische Strategie weiter entwickelt werden.

Drosophila melanogaster as a model to study host-parasitoid interactions: the case of the polydnaviral protein TnBVANK1

Parasitic wasps attack a number of insect species on which they feed, either externally or internally. This requires very effective strategies for suppressing the immune response and a finely tuned interference with the host physiology that is co-opted for the developing parasitoid progeny. The wealth of physiological host alterations is mediated by virulence factors encoded by the wasp or, in some cases, by polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition along with the egg, venom and ovarian secretions. PDVs are among the most powerful immunosuppressors in nature, targeting insect defense barriers at different levels. During my PhD research program I have used Drosophila melanogaster as a model to expand the functional analysis of virulence factors encoded by PDV focusing on the molecular processes underlying the disruption of the host endocrine system. I focused my research on a member of the ankyrin (ank) gene family, an immunosuppressant found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of ecdysteroid precursors in the cells of the prothoracic gland and results in developmental arrest.

Diagnosis and Treatment of Venous Malformations Consensus Document of the International Union of Phlebology (IUP): updated 2013

Venous malformations (VMs) are the most common vascular developmental anomalies (birth defects) . These defects are caused by developmental arrest of the venous system during various stages of embryogenesis. VMs remain a difficult diagnostic and therapeutic challenge due to the wide range of clinical presentations, unpredictable clinical course, erratic response to the treatment with high recurrence/persistence rates, high morbidity following non-specific conventional treatment, and confusing terminology. The Consensus Panel reviewed the recent scientific literature up to the year 2013 to update a previous IUP Consensus (2009) on the same subject. ISSVA Classification with special merits for the differentiation between the congenital vascular malformation (CVM) and vascular tumors was reinforced with an additional review on syndrome-based classification. A "modified" Hamburg classification was adopted to emphasize the importance of extratruncular vs. truncular sub-types of VMs. This incorporated the embryological origin, morphological differences, unique characteristics, prognosis and recurrence rates of VMs based on this embryological classification. The definition and classification of VMs were strengthened with the addition of angiographic data that determines the hemodynamic characteristics, the anatomical pattern of draining veins and hence the risk of complication following sclerotherapy. The hemolymphatic malformations, a combined condition incorporating LMs and other CVMs, were illustrated as a separate topic to differentiate from isolated VMs and to rectify the existing confusion with name-based eponyms such as Klippel-Trenaunay syndrome. Contemporary concepts on VMs were updated with new data including genetic findings linked to the etiology of CVMs and chronic cerebrospinal venous insufficiency. Besides, newly established information on coagulopathy including the role of D-Dimer was thoroughly reviewed to provide guidelines on investigations and anticoagulation therapy in the management of VMs. Congenital vascular bone syndrome resulting in angio-osteo-hyper/hypotrophy and (lateral) marginal vein was separately reviewed. Background data on arterio-venous malformations was included to differentiate this anomaly from syndrome-based VMs. For the treatment, a new section on laser therapy and also a practical guideline for follow up assessment were added to strengthen the management principle of the multidisciplinary approach. All other therapeutic modalities were thoroughly updated to accommodate a changing concept through the years.

Presence of polydnavirus transcripts in an egg-larval parasitoid and its lepidopterous host

The parasitoid Chelonus inanitus (Braconidae, Hymenoptera) oviposits into eggs of Spodoptera littoralis (Noctuidae, Lepidoptera) and, along with the egg, also injects polydnaviruses and venom, which are prerequisites for successful parasitoid development. The parasitoid larva develops within the embryonic and larval stages of the host, which enters metamorphosis precociously and arrests development in the prepupal stage. Polydnaviruses are responsible for the developmental arrest and interfere with the host's endocrine system in the last larval instar. Polydnaviruses have a segmented genome and are transmitted as a provirus integrated in the wasp's genome. Virions are only formed in female wasps and no virus replication is seen in the parasitized host. Here it is shown that very small amounts of viral transcripts were found in parasitized eggs and early larval instars of S. littoralis. Later on, transcript quantities increased and were highest in the late last larval instar for two of the three viral segments tested and in the penultimate to early last larval instar for the third segment. These are the first data on the occurrence of viral transcripts in the host of an egg-larval parasitoid and they are different from data reported for hosts of larval parasitoids, where transcript levels are already high shortly after parasitization. The analysis of three open reading frames by RT-PCR revealed viral transcripts in parasitized S. littoralis and in female pupae of C. inanitus, indicating the absence of host specificity. For one open reading frame, transcripts were also seen in male pupae, suggesting transcription from integrated viral DNA.