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

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

Levels of <i>O. dioica</i> E2F1 decline differentially in somatic endocycling cells versus mitotic germline cells during growth arrest.

<p>A) In somatic endocycling cells, E2F1 was constitutively present during both G- (arrows) and S-phases, under standard culture conditions. E2F1 was rapidly reduced in somatic endocycling cells during GA. This reduction coincided with a lack of incorporation of the S-phase marker IdU. B) Under standard culture conditions, E2F1 levels peaked during G1 (arrows) in mitotic germ line nuclei and were greatly reduced during S-phase. E2F1 levels persisted longer in the mitotic germline of GA animals than in somatic endocycling cells. Scale bars = 10 μm.</p

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 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

Inhibition of TOR signaling prevents meiotic onset in <i>Oikopleura dioica</i>.

<p>At standard culture densities (top row), the <i>O. dioica</i> germline is characterized by a coenocystic cytoplasm consisting of a homogenous population of proliferative mitotic nuclei prior to day 3 of development. Following meiotic onset, (late day 3) the germline coenocystic cytoplasm consists of equal numbers of smaller meiotic nuclei (MN, arrowheads) arrested in prophase I and larger endocycling nurse nuclei (NN). Germline nuclei of growth arrested animals (Dense condition) retain a homogeneous undifferentiated size and do not commence meiosis as evident from the persistence of the proliferative mitotic mark H3pS28 (arrows) similar to Day3 animals before meiotic onset under standard conditions. There was a decrease in IdU incorporation in germline nuclei over time (D3–D18; day 3 to day 18). Similarly, germ line nuclei of <i>O. dioica</i> cultured at standard density, do not commence meiosis when treated with the TOR inhibitor CCI-779 (7.5 μM) for 24 h, 48 h or 72 h and continue to proliferate mitotically. Scale bars = 20 μm.</p

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

Functional specialization of chordate CDK1 paralogs during oogenic meiosis

<p>Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, <i>Oikopleura dioica</i>, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, <i>O. dioica</i> has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program.</p

Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications

Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications