Alex McDougall

Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1

Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis. It is not clear which model best fits the mammalian system. Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1. Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC. These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles

In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. We demonstrate that the MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, we identified the network of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, we show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. These observations lead us to propose a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi.

Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors

Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10°/minute) and migrate (3 μm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin

Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).

Restaging the Spindle Assembly Checkpoint in Female Mammalian Meiosis I

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable forensuring the fidelity of chromosome segregation by delaying cell-cycle progression in theface of even a single misaligned chromosome. In contrast, the role of the SAC inunperturbed mammalian oocytes is less well defined as progression through meiosis I isunaltered in mouse oocytes in the presence of one or a few misaligned chromosomes.Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouseoocytes have produced conflicting results. To gain further insight into SAC functionduring female mammalian meiosis I, we recently utilised a morpholino-based antisenseapproach to deplete the majority of Mad2 in mouse oocytes. Our results define a clearrole for Mad2 in ensuring the proper timing of meiosis I events and ultimately, inensuring the fidelity of homologue disjunction. We discuss the implications of theseresults for the regulation of meiosis I in mammalian oocytes and for the genesis of humananeuploidy.

Fertilisation calcium signals in the ascidian egg.

Abstract The egg of ascidians (urochordate), as virtually all animal and plant species, displays Ca2+ signals upon fertilisation. These Ca2+ signals are repetitive Ca2+ waves that initiate in the cortex of the egg and spread through the whole egg interior. Two series of Ca2+ waves triggered from two distinct Ca2+ wave pacemakers entrain the two meiotic divisions preceding entry into the first interphase. The second messenger inositol (1,4,5) trisphosphate (IP3) is the main mediator of these global Ca2+ waves. Other Ca2+ signalling pathways (RyR and NAADPR) are functional in the egg but they mediate localised cortical Ca2+ signals whose physiological significance remains unclear. The meiosis I Ca2+ wave pacemaker is mobile and relies on intracellular Ca2+ release from the endoplasmic reticulum (ER) induced by a large production of IP3 at the sperm aster site. The meiosis II Ca2+ wave pacemaker is stably localised in a vegetal protrusion called the contraction pole. It is probable that a local production of IP3 in the contraction pole determines the site of this second pacemaker while functional interactions between ER and mitochondria regulate its activity. Finally, a third ectopic pacemaker can be induced by a global increase in IP3, making the ascidian egg a unique system where three different Ca2+ wave pacemakers coexist in the same cell.

Mos limits the number of meiotic divisions in urochordate eggs

Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca2+ oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.

Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes.

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cδ1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.

A novel mechanism controls the Ca2+ oscillations triggered by activation of ascidian eggs and has an absolute requirement for Cdk1 activity.

Fertilisation in ascidians triggers a series of periodic rises in cytosolic Ca2+ that are essential for release from metaphase I arrest and progression through meiosis II. These sperm-triggered Ca2+ oscillations are switched off at exit from meiosis II. Ascidian zygotes provided the first demonstration of the positive feedback loop whereby elevated Cdk1 activity maintained these Ca2+ oscillations. Since then it has been reported that Cdk1 sensitises the type I inositol trisphosphate [Ins(1,4,5)P3] receptor in somatic cells, and that sperm-triggered Ca2+ oscillations in mouse zygotes stop because the forming pronuclei sequester phospholipase C zeta that was delivered to the egg by the fertilising sperm. Here, using enucleation, we demonstrate in ascidian eggs that Ca2+ spiking stops at the correct time in the absence of pronuclei. Sequestration of sperm factor is therefore not involved in terminating Ca2+ spiking for these eggs. Instead we found that microinjection of the Cdk1 inhibitor p21 blocked Ca2+ spiking induced by ascidian sperm extract (ASE). However, such eggs were still capable of releasing Ca2+ in response to Ins(1,4,5)P3 receptor agonists, indicating that ASE-triggered Ca2+ oscillations can stop even though the response to Ins(1,4,5)P3 remained elevated. These data suggest that Cdk1 activity promotes Ins(1,4,5)P3 production in the presence of the sperm factor, rather than sensitising the Ca2+ releasing machinery to Ins(1,4,5)P3. These findings suggest a new link between this cell cycle kinase and the Ins(1,4,5)P3 pathway.