Histone variant innovation in a rapidly evolving chordate lineage

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Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics

<p>Abstract</p> <p>Background</p> <p>Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in <it>Oikopleura dioica </it>genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo).</p> <p>Results</p> <p><it>Oikopleura </it>genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways.</p> <p>Conclusions</p> <p><it>Oikopleura </it>has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.</p

OikoBase: a genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica

We report the development of OikoBase (http://oikoarrays.biology.uiowa.edu/Oiko/), a tiling array-based genome browser resource for Oikopleura dioica, a metazoan belonging to the urochordates, the closest extant group to vertebrates. OikoBase facilitates retrieval and mining of a variety of useful genomics information. First, it includes a genome browser which interrogates 1260 genomic sequence scaffolds and features gene, transcript and CDS annotation tracks. Second, we annotated gene models with gene ontology (GO) terms and InterPro domains which are directly accessible in the browser with links to their entries in the GO (http://www.geneontology.org) and InterPro (http://www.ebi.ac.uk/interpro/) databases, and we provide transcript and peptide links for sequence downloads. Third, we introduce the transcriptomics of a comprehensive set of developmental stages of O. dioica at high resolution and provide download- able gene expression data for all developmental stages. Fourth, we incorporate a BLAST tool to identify homologs of genes and proteins. Finally, we include a tutorial that describes how to use OikoBase as well as a link to detailed methods, explaining the data generation and analysis pipeline. OikoBase will provide a valuable resource for research in chordate development, genome evolution and plasticity and the molecular ecology of this important marine planktonic organism.publishedVersio

Lifespan extension in a semelparous chordate occurs via developmental growth arrest just prior to meiotic entry

It is proposed that the ageing process is linked to signaling from the germline such that the rate of ageing can be adjusted to the state of the reproductive system, allowing these two processes to co-evolve. Mechanistic insight into this link has been primarily derived from iteroparous reproductive models, the nematode C. elegans, and the arthropod Drosophila. Here, we examined to what extent these mechanisms are evolutionarily conserved in a semelparous chordate, Oikopleura dioica, where we identify a developmental growth arrest (GA) in response to crowded, diet-restricted conditions, which can extend its lifespan at least three-fold. Under nutritional stress, the iteroparative models sacrifice germ cells that have entered meiosis, while maintaining a reduced pool of active germline stem cells (GSCs). In contrast, O. dioica only entered GA prior to meiotic entry. Stress conditions encountered after this point led to maturation in a normal time frame but with reduced reproductive output. During GA, TOR signaling was inhibited, whereas MAPK, ERK1/2 and p38 pathways were activated, and under such conditions, activation of these pathways was shown to be critical for survival. Direct inhibition of TOR signaling alone was sufficient to prevent meiotic entry and germline differentiation. This inhibition activated the p38 pathway, but did not activate the ERK1/2 pathway. Thus, the link between reproductive status and lifespan extension in response to nutrientlimited conditions is interpreted in a significantly different manner in these iteroparative versus semelparous models. In the latter case, meiotic entry is a definitive signal that lifespan extension can no longer occur, whereas in the former, meiotic entry is not a unique chronological event, and can be largely erased during lifespan extension in response to nutrient stress, and reactivated from a pool of maintained GSCs when conditions improve

Switching of INCENP paralogs controls transitions in mitotic chromosomal passenger complex functions

A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, Oikopleura dioica, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate O. dioica

Inhibition of TOR signaling or dietary restriction mimics growth arrest in <i>Oikopleura dioica.</i>

<p>A) When <i>O. dioica</i> was cultured at standard densities in the 24 h presence of the TOR inhibitor CCI-779 (7.5 μM) or 24 h in the absence of food, a significant (**p<0.01) growth arrest (GA) similar to that identified under dense conditions was observed. B) When animals were cultured under dense conditions, GA was alleviated by increasing food availability in a dose-dependent manner. Significant differences (**p<0.01, ***p<0.001) in GA and maturation at 2X and 4X diets are indicated with respect to those animals maintained at a 1X diet under dense conditions. Error bars in A and B indicate standard errors. C) <i>O. dioica</i> cultured under standard conditions in the presence of 7.5 μM TOR inhibitor CCI-779 (rapamycin analog) for 24 h or animals cultured without food for 24 h also showed arrest of somatic endocycles whereas mitotically proliferating germ nuclei continued to cycle. Scale bars = 50 μm in A and B. D) Western blots showed that TOR signaling was inhibited in GA animals cultured under dense conditions as it was in animals cultured at standard densities in the presence of the TOR inhibitor CCI-779. Activation of the downstream effectors of TOR signaling, 4EBP1 and RPS6, was reduced under both of these treatments as compared to animals cultured at standard densities, in the presence or absence of DMSO.</p

Growth arrest only occurs prior to meiotic entry.

<p>A) <i>O. dioica</i> only undergoes GA if exposed to dense, nutrient-limited conditions prior to day 4 of development under standard conditions. The schema at the left summarizes the experimental design. Animals were cultured under standard conditions and then introduced to dense culture conditions at either day 3, 4, or 5. GA was very significantly enhanced in those experiencing dense conditions prior to day 4 (Dense D3 ) compared to those exposed from day 4 or 5 (***p<0.001, with respect to normally developing animals at standard density). Maturation was significantly reduced in animals exposed to increased density prior to day 4 whereas those exposed later matured with the same timing as animals maintained throughout the experiment under standard culture conditions. B) Animals released from GA by dilution to standard density complete maturation and spawning in 2.5–3.5 days, a time frame similar to that required for animals with a day 3 morphology cultured under standard conditions. The fecundity of animals released from GA was not significantly affected compared to animals cultured under standard conditions throughout, whereas in contrast, animals cultured initially under standard conditions and then transferred to higher density at day 4 or 5, exhibited significantly reduced fecundity (**p<0.01). C) In addition to reduced fecundity, there was also a slight reduction (*p<0.05) in the quality of the eggs produced by animals exposed to dense conditions from day 4 or 5 as judged by their capacity to generate embryos that developed and hatched successfully with normal morphology. The schema shown below panels B and C depicts the experimental design corresponding to both of these result panels. D) Inhibition of TOR signaling in the presence of CCI-779 was more efficient in establishing GA and preventing animal maturation when introduced from day 3 onwards (D3 ), prior to initiation of meiosis in females, than it was when introduced from day 4 onwards, after meiotic entry. Significant differences in GA and maturation are indicated with respect to animals cultured at standard density in the presence of DMSO from day 3 onwards. Error bars indicate standard error.</p

Growth arrest in <i>Oikopleura dioica</i>.

<p>A) At standard densities <i>O. dioica</i> completes its life cycle in 6 days at which point it spawns and dies. At higher culture densities <i>O. dioica</i> undergoes growth arrest (GA) and the animal can exhibit a 3-fold increase in lifespan, albeit, with increasing mortality up to day 18. B) At high culture densities GA animals undergo very limited increase in body length compared to control animals at standard culture densities (significant differences: *p<0.05, **p<0.01). The morphology of surviving GA animals throughout the 18–day period remained similar to that of day 3 animals cultured under standard conditions. Error bars indicate standard errors in A and B. C) A rapid post-day 3 reduction in IdU incorporation (S phase marker) was observed in somatic endocycling cells in animals cultured under dense conditions. IdU incorporation persisted longer in mitotically proliferating germ nuclei in these same animals but eventually diminished as growth arrest persisted (D3 <i>d</i> = day 3 dense etc.). Scale bars = 50 μm.</p

Following release from growth arrest, mitotic germline nuclei re-enter S-phase before somatic endocycling cells.

<p>A) IdU (S-phase marker) incorporation was gradually restored in the endocycling epithelial and mitotic germ line nuclei of GA animals that had been released into standard culture conditions. Scale bars = 50 μm. B) Pulse chases of CldU and IdU (see also materials and methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093787#pone.0093787.s003" target="_blank">Fig. S3</a>) in GA animals (top panels), or in animals that had been released from GA for 2 (mid-panels) or 3 h (bottom panels), revealed that resumption of S-phase in the mitotic germline preceded that in somatic endocycling cells by 1 h. Scale bars = 50 μm. Insets depict zooms of the germline (Scale bars = 10 μm).</p