The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation.

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.

Peripheral astral microtubules ensure asymmetric furrow positioning in neural stem cells.

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.

Drosophila Tubulin-Specific Chaperone E Recruits Tubulin around Chromatin to Promote Mitotic Spindle Assembly.

Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. In this study, we found that the Drosophila tubulin-specific chaperone dTBCE is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. Our data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. We propose that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.

Dual control of Kinesin-1 recruitment to microtubules by Ensconsin in Drosophila neuroblasts and oocytes.

Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro We propose that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.

Drosophila Aurora A regulates mitotic timing in cancer stem cells: Possible therapeutic implications.

Loss of Aurora A in Drosophila neuroblasts promotes loss of cell fate, leading to brain tumors. We showed that these tumor stem cells are delayed during mitosis and efficiently segregate their chromosomes even without the spindle assembly checkpoint. Here, we discuss the possible relevance of our results to human cancers.

Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila.

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells.

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

Oocyte-somatic cells interactions, lessons from evolution.

BACKGROUND: Despite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates. RESULTS: Using a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis. CONCLUSIONS: Our study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg.

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition.

Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes.

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5'monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-L-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.

EP45 accumulates in growing Xenopus laevis oocytes and has oocyte-maturation-enhancing activity involved in oocyte quality.

The capacity of oocytes to fully support meiotic maturation develops gradually during oocyte growth. Growing oocytes accumulate proteins and mRNAs required for this process. However, little is known about the identity of these factors. We performed a differential proteomic screen comparing the proteomes of growing stage-IV oocytes, which do not undergo meiotic maturation in response to progesterone, with fully grown stage-VI ones, which do. In 2D gels of stage-VI oocytes, we identified a group of four protein spots as EP45 (estrogen-regulated protein 45 kDa), which belongs to the family of serine protease inhibitors and is also known as Seryp or pNiXa. Western blot analysis after mono- and bi-dimensional electrophoreses confirmed the accumulation of certain forms of this protein in oocytes between stages IV and VI. EP45 mRNA was not detectable in oocytes or ovaries, but was expressed in the liver. A low-mobility isoform of EP45 was detected in liver and blood, whereas two (occasionally three or four) higher-mobility isoforms were found exclusively in oocytes, suggesting that liver-synthesized protein is taken up by oocytes from the blood and rapidly modified. Alone, overexpression of RNA encoding either full-length or N-terminally truncated protein had no effect on meiotic resumption in stage-IV or -VI oocytes. However, in oocytes moderately reacting to low doses of progesterone, it significantly enhanced germinal-vesicle breakdown, showing a novel and unsuspected activity of this protein. Thus, EP45 accumulates in growing oocytes through uptake from the blood and has the capacity to act as an 'oocyte-maturation enhancer' ('Omen').

Role of oocyte quality in meiotic maturation and embryonic development.

The quality of oocytes plays a key role in a proper embryo development. In humans, oocytes of poor quality may be the cause of women infertility and an important obstacle in successful in vitro fertilization (IVF). The competence of oocytes depends on numerous processes taking place during the whole oogenesis, but its final steps such as oocyte maturation, seem to be of key importance. In this paper, we overview factors involved in the development of a fully functional female gamete with Xenopus laevis as a major experimental model. Modern approaches, e.g. proteomic analysis, enable the identification of novel proteins involved in oocyte development. EP45, called also Seryp or pNiXa, which belongs to the serpin (serine protease inhibitors) super-family is one of such recently analyzed proteins. This protein seems to be involved in the stimulation of meiotic maturation and embryo development. EP45 is potentially a key factor in correct oocyte development and determining the quality of oocytes.

Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development.

ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N-cam expression patterns, whereas krox-20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos-7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.

ZFPIP/Zfp462 is maternally required for proper early Xenopus laevis development.

ZFPIP (Zinc Finger Pbx1 Interacting Protein) has been recently identified in our laboratory in a yeast two hybrid screen using an embryonic mouse cDNA library and PBX1 as a bait. This gene encodes a large protein (250 kDa) that contains a bipartite NLS, numerous C2H2 zinc fingers and is highly conserved amongst vertebrates. In order to address the role of ZFPIP during embryonic development, we analysed the expression pattern of the gene and performed morpholinos injections into Xenopus laevis embryos. We first showed that the ZFPIP protein was maternally present in oocytes. Then, ZFPIP was detected from morula to neurula stages in the nucleus of the cells, with a gradient from animal to vegetal pole. By injection of ZFPIP morpholinos, we showed that morphant embryos were unable to undergo proper gastrulation and subsequently exhibited a persistent opened blastopore. Analysis of molecular and cellular events that were altered in morphant embryos highlighted an impairment of cell division processes as illustrated by atypical mitosis with aberrant metaphase, anaphase or telophase, incomplete chromosome segregation or conjointed nuclei. The overall data presented here demonstrated that ZFPIP was a major developing gene that acts in the very first steps of embryonic development of Xenopus laevis.

Temporal regulation of embryonic M-phases.

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.

Temporal regulation of the first mitosis in Xenopus and mouse embryos.

Cell cycle regulation in Eukaryotes is based on common molecular actors and mechanisms. However, the canonical cell cycle is modified in certain cells. Such modifications play a key role in oocyte maturation and embryonic development. They can be achieved either by introduction of new components, pathways, substrates, changed interactions between them, or by elimination of some factors inherited by the cells from previous developmental stages. Here we discuss a particular temporal regulation of the first embryonic M-phase of Xenopus and mouse embryos. These two examples help to understand better the general regulation of M-phase of the cell cycle.

Specific expression of olfactory binding protein in the aerial olfactory cavity of adult and developing Xenopus.

Olfactory binding proteins (OBP), commonly associated with aerial olfaction, are found in the olfactory mucus of mammals but have never been identified in fish. It is still not clear whether the presence of OBP in aerial olfactory systems is due to phylogenetic or to functional differences linked to the adaptation of the olfactory system to an aerial environment. To test this alternative, the olfactory system of Xenopus offers a unique opportunity because it includes two olfactory cavities, one of which is thought to be devoted to aquatic olfaction and the other to aerial olfaction. We therefore purified and cloned OBPs in two Xenopus species. Xenopus laevis OBP (XlaeOBP) and Xenopus tropicalis OBP (XtroOBP) exhibit 158 and 160 amino acids, respectively, sharing 89 residues. cRNA probes allowed us to demonstrate that XlaeOBP and XtroOBP are expressed at the level of Bowman's gland specifically in the aerial olfactory cavity, as confirmed using anti-XlaeOBP antiserum. OBP mRNA transcription occurs early during metamorphosis, as early as stage 57. This is the first study to demonstrate that OBPs are exclusively present in the aerial chamber and are only expressed as the tadpole becomes an adult in species which possess both aquatic and aerial olfactory organs.

Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts.

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3.

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.

G1/S phase cyclin-dependent kinase overexpression perturbs early development and delays tissue-specific differentiation in Xenopus.

Cell division and differentiation are largely incompatible but the molecular links between the two processes are poorly understood. Here, we overexpress G1/S phase cyclins and cyclin-dependent kinases in Xenopus embryos to determine their effect on early development and differentiation. Overexpression of cyclin E prior to the midblastula transition (MBT), with or without cdk2, results in a loss of nuclear DNA and subsequent apoptosis at early gastrula stages. By contrast, overexpressed cyclin A2 protein does not affect early development and, when stabilised by binding to cdk2, persists to tailbud stages. Overexpression of cyclin A2/cdk2 in post-MBT embryos results in increased proliferation specifically in the epidermis with concomitant disruption of skin architecture and delay in differentiation. Moreover, ectopic cyclin A2/cdk2 also inhibits differentiation of primary neurons but does not affect muscle. Thus, overexpression of a single G1/S phase cyclin/cdk pair disrupts the balance between division and differentiation in the early vertebrate embryo in a tissue-specific manner.