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Hiiragi GroupPublications

Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.
Ohnishi, Y., Huber, W., Tsumura, A., Kang, M., Xenopoulos, P., Kurimoto, K., Oles, A.K., Arauzo-Bravo, M.J., Saitou, M., Hadjantonakis, A.K. & Hiiragi, T.
Nat Cell Biol. 2014 Jan;16(1):27-37. doi: 10.1038/ncb2881. Epub 2013 Dec 1.
It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.
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A self-organization framework for symmetry breaking in the mammalian embryo.
Wennekamp, S., Mesecke, S., Nedelec, F. & Hiiragi, T.
Nat Rev Mol Cell Biol. 2013 Jul;14(7):452-9. doi: 10.1038/nrm3602. Epub 2013 Jun19.
The mechanisms underlying the appearance of asymmetry between cells in the early embryo and consequently the specification of distinct cell lineages during mammalian development remain elusive. Recent experimental advances have revealed unexpected dynamics of and new complexity in this process. These findings can be integrated in a new unified framework that regards the early mammalian embryo as a self-organizing system.
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The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development.
Courtois, A., Schuh, M., Ellenberg, J. & Hiiragi, T.
J Cell Biol. 2012 Aug 6;198(3):357-70. Epub 2012 Jul 30.
The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12-, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo.
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The Kruppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo.
Groner, A.C., Tschopp, P., Challet, L., Dietrich, J.E., Verp, S., Offner, S., Barde, I., Rodriguez, I., Hiiragi, T. & Trono, D.
J Biol Chem. 2012 Jul 20;287(30):25361-9. Epub 2012 May 17.
The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Kruppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.
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Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development.
Lavial, F., Bessonnard, S., Ohnishi, Y., Tsumura, A., Chandrashekran, A., Fenwick, M.A., Tomaz, R.A., Hosokawa, H., Nakayama, T., Chambers, I., Hiiragi, T., Chazaud, C. & Azuara, V.
Genes Dev. 2012 Jul 1;26(13):1445-58. Epub 2012 Jun 19.
The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.
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Stochastic processes in the development of pluripotency in vivo.
Wennekamp, S. & Hiiragi, T.
Biotechnol J. 2012 Jun;7(6):737-44. doi: 10.1002/biot.201100357. Epub 2012 Apr27.
The divergence of the pluripotent inner cell mass and extraembryonic trophectoderm from an apparently homogenous population of cells is a decisive event in mammalian preimplantation development. While three models have been proposed to explain early cellular differentiation in the mouse embryo, the initial cue generating asymmetry within the embryo remains elusive. Recently, unexpected heterogeneity in the expression of crucial transcription factors within the blastocyst has raised the intriguing possibility that a stochastic component is involved in lineage divergence. Unraveling the molecular dynamics and developmental function of the observed heterogeneity awaits further investigations at the single-cell level using quantitative live-imaging with appropriate reporter lines. The possible involvement of dynamic heterogeneity in the establishment, maintenance and resolution of pluripotency makes this topic highly relevant not only to developmental biology, but also to stem cell research and regenerative medicine. In this review, we discuss the possible involvement of stochastic processes in lineage divergence and the establishment of pluripotency in vivo, based on recent data from mouse embryology and stem cell research.
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Computer simulation of emerging asymmetry in the mouse blastocyst.
Honda, H., Motosugi, N., Nagai, T., Tanemura, M. & Hiiragi, T.
Development. 2008 Apr;135(8):1407-14.
The mechanism of embryonic polarity establishment in mammals has long been controversial. Whereas some claim prepatterning in the egg, we recently presented evidence that mouse embryonic polarity is not established until blastocyst and proposed the mechanical constraint model. Here we apply computer simulation to clarify the minimal cellular properties required for this morphology. The simulation is based on three assumptions: (1) behavior of cell aggregates is simulated by a 3D vertex dynamics model; (2) all cells have equivalent mechanical properties; (3) an inner cavity with equivalent surface properties is gradually enlarged. However, an initial attempt reveals a requirement for an additional assumption: (4) the surface of the cavity is firmer than intercellular surfaces, suggesting the presence of a basement membrane lining the blastocyst cavity, which is indeed confirmed by published data. The simulation thus successfully produces a structure recapitulating the mouse blastocyst. The axis of the blastocyst, however, remains variable, leading us to an additional assumption: (5) the aggregate is enclosed by a capsule, equivalent to the zona pellucida in vivo. Whereas a spherical capsule does not stabilize the blastocyst axis, an ellipsoidal capsule eventually orients the axis in accordance with its longest diameter. These predictions are experimentally verified by time-lapse recordings of mouse embryos. During simulation, equivalent cells form two distinct populations composed of smaller inner cells and larger outer cells. These results reveal a unique feature of early mammalian development: an asymmetry may emerge autonomously in an equivalent population with no need for a priori intrinsic differences.
Europe PMC

Dynamic rearrangement of surface proteins is essential for cytokinesis.
Bauer, T., Motosugi, N., Miura, K., Sabe, H. & Hiiragi, T.
Genesis. 2008 Mar;46(3):152-62. doi: 10.1002/dvg.20377.
Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.
Europe PMC

Hypomethylation of paternal DNA in the late mouse zygote is not essential for development.
Polanski, Z., Motosugi, N., Tsurumi, C., Hiiragi, T. & Hoffmann, S.
Int J Dev Biol. 2008;52(2-3):295-8.
Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.
Europe PMC

Stochastic processes during mouse blastocyst patterning.
Dietrich, J.E. & Hiiragi, T.
Cells Tissues Organs. 2008;188(1-2):46-51. Epub 2008 Feb 27.
Mammalian preimplantation development serves to form a blastocyst, a structure that is able to implant into the mother's uterus to support further development of the embryo proper. In the developing conceptus, the first differentiation events separate the epithelial trophectoderm from the inner cell mass, which is comprised of the primitive endoderm and embryo-generating epiblast. Although the process of blastocyst formation may appear simple, its morphogenesis and the mechanism(s) of lineage specification are not yet fully understood. Here we discuss findings that suggest the involvement of stochastic processes and the influence of external cues in the patterning process during mouse preimplantation development.
Europe PMC

Stochastic patterning in the mouse pre-implantation embryo.
Dietrich, J.E. & Hiiragi, T.
Development. 2007 Dec;134(23):4219-31. Epub 2007 Oct 31.
Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, we were able to identify critical events leading to the divergence of TE, EPI and PE lineages. We found that Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, we propose a model in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development.
Europe PMC

Embryology: does prepatterning occur in the mouse egg?
Hiiragi, T., Louvet-Vallee, S., Solter, D. & Maro, B.
Nature. 2006 Jul 13;442(7099):E3-4; discussion E4.
A recurring question in developmental biology has been whether localized determinants play any role in mammalian preimplantation development. This is a controversial issue that brings back the idea of prepatterning and is explored further by Plusa et al., who claim it is the first cleavage of the mouse zygote that predicts the blastocyst axis, rather than the animal pole or sperm entry point, as previously suggested. However, other evidence indicates that the blasotcyst axis is not predetermined and there is no prepatterning in the mouse egg. Here we investigate the origin of these different views and conclude that they arise from differences in the data themselves and in their interpretation.
Europe PMC

Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte.
Motosugi, N., Dietrich, J.E., Polanski, Z., Solter, D. & Hiiragi, T.
PLoS Biol. 2006 May;4(5):e135. Epub 2006 Apr 25.
Knowledge about the mechanism that establishes embryonic polarity is fundamental in understanding mammalian development. In re-addressing several controversial claims, we recently proposed a model in which mouse embryonic polarity is not specified until the blastocyst stage. Before fertilization, the fully differentiated oocyte has been characterized as "polarized," and we indeed observed that the sperm preferentially enters the polar body half. Here we show that preferential sperm entry is not due to an intrinsic polarity of the oocyte, since fertilization takes place uniformly when the zona pellucida is removed. We suggest that the term "asymmetry" denotes morphological differences, whereas "polarity" in addition implies developmental consequences. Thus, the mouse oocyte can be considered "asymmetric" but "non-polarized." The penetration through the zona pellucida is also random, and a significant proportion of sperm binds to the oocyte membrane at a point distant from the zona penetration site. Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity.
Europe PMC

Fatal flaws in the case for prepatterning in the mouse egg.
Hiiragi, T. & Solter, D.
Reprod Biomed Online. 2006 Feb;12(2):150-2.
The presence or absence of predetermination and polarity in the mouse preimplantation embryo is still controversial. The question is if the mechanisms underlying early mammalian development is comparable to those operating in non-mammalian 'model' organisms. In a recent article by Gardner in this journal, the author criticizes two of our recent publications. However, in order to resolve this controversy it is essential to read relevant reports carefully without bias and to provide data on which a particular claim is based.
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Polarity of the mouse embryo is established at blastocyst and is not prepatterned.
Motosugi, N., Bauer, T., Polanski, Z., Solter, D. & Hiiragi, T.
Genes Dev. 2005 May 1;19(9):1081-92.
Polarity formation in mammalian preimplantation embryos has long been a subject of controversy. Mammalian embryos are highly regulative, which has led to the conclusion that polarity specification does not exist until the blastocyst stage; however, some recent reports have now suggested polarity predetermination in the egg. Our recent time-lapse recordings have demonstrated that the first cleavage plane is not predetermined in the mouse egg. Here we show that, in contrast to previous claims, two-cell blastomeres do not differ and their precise future contribution to the inner cell mass and/or the trophectoderm cannot be anticipated. Thus, all evidence so far strongly suggests the absence of predetermined axes in the mouse egg. We observe that the ellipsoidal zona pellucida exerts mechanical pressure and
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Mechanism of first cleavage specification in the mouse egg: is our body plan set at day 0?
Hiiragi, T. & Solter, D.
Cell Cycle. 2005 May;4(5):661-4. Epub 2005 May 22.
In most animals the body axis is specified in the egg. Because of their highly regulative capacity after experimental manipulations, mammalian preimplantation embryos have long been thought to be an exception to this rule, lacking polarity until the blastocyst stage. However, it has recently been suggested that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst arises perpendicular to the first cleavage plane. Considering the second polar body (2pb) as a stationary marker for the "animal pole (A-pole)" during preimplantation development, the authors concluded that the polarity of the mouse embryo is already specified in the egg, as is the case for most non-mammalian animals. However, the results of our recent time-lapse recordings have shown(8) that in 50% of the embryos the first cleavage occurs at a considerable distance from the "animal-vegetal (A-V) axis" and that the 2pb moves towards the first cleavage plane, in contrast to the previous claims. Thus, there is no predetermined axis in the mouse egg. We also presented a novel model for specification of the first cleavage plane: this is defined as the plane separating the two apposing pronuclei that have moved to the center of the egg. In this review we will elucidate the discrepancy between the previous model and our model, and discuss the possible causes.
Europe PMC

First cleavage plane of the mouse egg is not predetermined but defined by the topology of the two apposing pronuclei.
Hiiragi, T. & Solter, D.
Nature. 2004 Jul 15;430(6997):360-4.
Studies of experimentally manipulated embryos have led to the long-held conclusion that the polarity of the mouse embryo remains undetermined until the blastocyst stage. However, recent studies reporting that the embryonic-abembryonic axis of the blastocyst arises perpendicular to the first cleavage plane, and hence to the animal-vegetal axis of the zygote, have led to the claim that the axis of the mouse embryo is already specified in the egg. Here we show that there is no specification of the axis in the egg. Time-lapse recordings show that the second polar body does not mark a stationary animal pole, but instead, in half of the embryos, moves towards a first cleavage plane. The first cleavage plane coincides with the plane defined by the two apposing pronuclei once they have moved to the centre of the egg. Pronuclear transfer experiments confirm that the first cleavage plane is not determined in early interphase but rather is specified by the newly formed topology of the two pronuclei. The microtubule networks that allow mixing of parental chromosomes before dividing into two may be involved in these processes.
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