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Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.

This corrects the article DOI: 10.1038/nbt.4015.

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro . Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis., (© 2017 Bury et al.)

Establishment of cell polarity in the mammalian embryo is fundamental for the first cell fate decision that sets aside progenitor cells for both the new organism and the placenta. Yet the sequence of events and molecular mechanism that trigger this process remain unknown. Here, we show that de novo polarisation of the mouse embryo occurs in two distinct phases at the 8-cell stage. In the first phase, an apical actomyosin network is formed. This is a pre-requisite for the second phase, in which the Par complex localises to the apical domain, excluding actomyosin and forming a mature apical cap. Using a variety of approaches, we also show that phospholipase C-mediated PIP 2 hydrolysis is necessary and sufficient to trigger the polarisation of actomyosin through the Rho-mediated recruitment of myosin II to the apical cortex. Together, these results reveal the molecular framework that triggers de novo polarisation of the mouse embryo.The molecular trigger that establishes cell polarity in the mammalian embryo is unclear. Here, the authors show that de novo polarisation of the mouse embryo at the 8-cell stage is directed by Phospholipase C and Protein kinase C and occurs in two phases: polarisation of actomyosin followed by the Par complex.

The correct temporal regulation of mitosis underpins genomic stability because it ensures the alignment of chromosomes on the mitotic spindle that is required for their proper segregation to the two daughter cells. Crucially, sister chromatid separation must be delayed until all the chromosomes have attached to the spindle; this is achieved by the Spindle Assembly Checkpoint (SAC) that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. In many species the first embryonic M-phase is significantly prolonged compared to the subsequent divisions, but the reason behind this has remained unclear. Here, we show that the first M-phase in the mouse embryo is significantly extended due to a delay in APC/C activation. Unlike in somatic cells, where the APC/C first targets cyclin A2 for degradation at nuclear envelope breakdown (NEBD), we find that in zygotes cyclin A2 remains stable for a significant period of time after NEBD. Our findings that the SAC prevents cyclin A2 degradation, whereas over-expressed Plk1 stimulates it, support our conclusion that the delay in cyclin A2 degradation is caused by low APC/C activity. As a consequence of delayed APC/C activation cyclin B1 stability in the first mitosis is also prolonged, leading to the unusual length of the first M-phase.

The separation of embryonic from extra-embryonic tissues within the inner cell mass to generate the epiblast (EPI), which will form the new organism, from the primitive endoderm (PE), which will form the yolk sac, is a crucial developmental decision. Here, we identify a chromatin modifier, Satb1, with a distinct role in this decision. Satb1 is differentially expressed within 16-cell-stage embryos, with higher expression levels in the inner cell mass progenitor cells. Depleting Satb1 increases the number of EPI cells at the expense of PE. This phenotype can be rescued by simultaneous depletion of both Satb1 and Satb2, owing to their antagonistic effect on the pluripotency regulator Nanog. Consequently, increasing Satb1 expression leads to differentiation into PE and a decrease in EPI, as a result of the modulation of expression of several pluripotency- and differentiation-related genes by Satb1. Finally, we show that Satb1 is a downstream target of the Fgf signalling pathway, linking chromatin modification and Fgf signalling. Together, these results identify a role for Satb1 in the lineage choice between pluripotency and differentiation and further our understanding of early embryonic lineage segregation., (© 2017. Published by The Company of Biologists Ltd.)

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos., (Copyright © 2017, American Association for the Advancement of Science.)

Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells-blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

Dynamic control of gene expression is essential for the development of a totipotent zygote into an embryo with defined cell lineages. The accessibility of genes responsible for cell specification to transcriptional machinery is dependent on chromatin remodelling complexes such as the SWI\SNF (BAF) complex. However, the role of the BAF complex in early mouse development has remained unclear. Here, we demonstrate that BAF155, a major BAF complex subunit, regulates the assembly of the BAF complex in vivo and regulates lineage specification of the mouse blastocyst. We find that associations of BAF155 with other BAF complex subunits become enriched in extra-embryonic lineages just prior to implantation. This enrichment is attributed to decreased mobility of BAF155 in extra-embryonic compared with embryonic lineages. Downregulation of BAF155 leads to increased expression of the pluripotency marker Nanog and its ectopic expression in extra-embryonic lineages, whereas upregulation of BAF155 leads to the upregulation of differentiation markers. Finally, we show that the arginine methyltransferase CARM1 methylates BAF155, which differentially influences assembly of the BAF complex between the lineages and the expression of pluripotency markers. Together, our results indicate a novel role of BAF-dependent chromatin remodelling in mouse development via regulation of lineage specification., (© 2016. Published by The Company of Biologists Ltd.)

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Specification of cell fate in the early mouse embryo involves the generation of heterogeneities between cells in polarity, position, gene expression, and protein activity. Identifying when and how these differences first become established represents an important challenge for understanding the roles of stochastic and cell history-dependent mechanisms in mammalian development., (© 2016 Elsevier Inc. All rights reserved.)

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development., (© 2015 The Authors.)

Understanding the past is to understand the present. Mammalian life, with all its complexity comes from a humble beginning of a single fertilized egg cell. Achieving this requires an enormous diversification of cellular function, the majority of which is generated through a series of cellular decisions during embryogenesis. The first decisions are made as the embryo prepares for implantation, a process that will require specialization of extra-embryonic lineages while preserving an embryonic one. In this mini-review, we will focus on the mouse as a mammalian model and discuss recent advances in the decision making process of the early embryo., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage., (© 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.)

The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which gives rise to the pluripotent epiblast and therefore the future embryo, and the trophectoderm (TE), which will build the placenta. The TE lineage depends on the transcription factor Cdx2. However, when Cdx2 first starts to act remains unclear. Embryos with zygotic deletion of Cdx2 develop normally until the late blastocyst stage leading to the conclusion that Cdx2 is important for the maintenance but not specification of the TE. In contrast, down-regulation of Cdx2 transcripts from the early embryo stage results in defects in TE specification before the blastocyst stage. Here, to unambiguously address at which developmental stage Cdx2 becomes first required, we genetically deleted Cdx2 from the oocyte stage using a Zp3-Cre/loxP strategy. Careful assessment of a large cohort of Cdx2 maternal-zygotic null embryos, all individually filmed, examined and genotyped, reveals an earlier lethal phenotype than observed in Cdx2 zygotic null embryos that develop until the late blastocyst stage. The developmental failure of Cdx2 maternal-zygotic null embryos is associated with cell death and failure of TE specification, starting at the morula stage. These results indicate that Cdx2 is important for the correct specification of TE from the morula stage onwards and that both maternal and zygotic pools of Cdx2 are required for correct pre-implantation embryogenesis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Pre-implantation development requires the specification and organization of embryonic and extra-embryonic lineages. The separation of these lineages takes place when asymmetric divisions generate inside and outside cells that differ in polarity, position and fate. Here we assess the global transcriptional identities of these precursor cells to gain insight into the molecular mechanisms regulating lineage segregation. Unexpectedly, this reveals that complementary components of the bone morphogenetic protein (BMP) signalling pathway are already differentially expressed after the first wave of asymmetric divisions. We investigate the role of BMP signalling by expressing dominant negative forms of Smad4 and Bmpr2, by downregulating the pathway using RNA interference against BMP ligands and by applying three different BMP inhibitors at distinct stages. This reveals that BMP signalling regulates the correct development of both extra-embryonic lineages, primitive endoderm and trophectoderm, but not the embryonic lineage, before implantation. Together, these findings indicate multiple roles of BMP signalling in the early mouse embryo.

A critical point in mammalian development is when the early embryo implants into its mother's uterus. This event has historically been difficult to study due to the fact that it occurs within the maternal tissue and therefore is hidden from view. In this review, we discuss how the mouse embryo is prepared for implantation and the molecular mechanisms involved in directing and coordinating this crucial event. Prior to implantation, the cells of the embryo are specified as precursors of future embryonic and extra-embryonic lineages. These preimplantation cell fate decisions rely on a combination of factors including cell polarity, position and cell-cell signalling and are influenced by the heterogeneity between early embryo cells. At the point of implantation, signalling events between the embryo and mother, and between the embryonic and extraembryonic compartments of the embryo itself, orchestrate a total reorganization of the embryo, coupled with a burst of cell proliferation. New developments in embryo culture and imaging techniques have recently revealed the growth and morphogenesis of the embryo at the time of implantation, leading to a new model for the blastocyst to egg cylinder transition. In this model, pluripotent cells that will give rise to the fetus self-organize into a polarized three-dimensional rosette-like structure that initiates egg cylinder formation.

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most - but not all - cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

Transformation of pluripotent epiblast cells into a cup-shaped epithelium as the mouse blastocyst implants is a poorly understood and yet key developmental step. Studies of morphogenesis in embryoid bodies led to the current belief that it is programmed cell death that shapes the epiblast. However, by following embryos developing in vivo and in vitro, we demonstrate that not cell death but a previously unknown morphogenetic event transforms the amorphous epiblast into a rosette of polarized cells. This transformation requires basal membrane-stimulated integrin signaling that coordinates polarization of epiblast cells and their apical constriction, a prerequisite for lumenogenesis. We show that basal membrane function can be substituted in vitro by extracellular matrix (ECM) proteins and that ES cells can be induced to form similar polarized rosettes that initiate lumenogenesis. Together, these findings lead to a completely revised model for peri-implantation morphogenesis in which ECM triggers the self-organization of the embryo's stem cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Lineage specification in the preimplantation mouse embryo is a regulative process. Thus, it has been difficult to ascertain whether segregation of the inner-cell-mass (ICM) into precursors of the pluripotent epiblast (EPI) and the differentiating primitive endoderm (PE) is random or influenced by developmental history. Here, our results lead to a unifying model for cell fate specification in which the time of internalization and the relative contribution of ICM cells generated by two waves of asymmetric divisions influence cell fate. We show that cells generated in the second wave express higher levels of Fgfr2 than those generated in the first, leading to ICM cells with varying Fgfr2 expression. To test whether such heterogeneity is enough to bias cell fate, we upregulate Fgfr2 and show it directs cells towards PE. Our results suggest that the strength of this bias is influenced by the number of cells generated in the first wave and, mostly likely, by the level of Fgf signalling in the ICM. Differences in the developmental potential of eight-cell- and 16-cell-stage outside blastomeres placed in the inside of chimaeric embryos further support this conclusion. These results unite previous findings demonstrating the importance of developmental history and Fgf signalling in determining cell fate.

Although in recent years we have seen an undeniable improvement in the field of reproductive biology and medicine, the efficiency of in vitro fertilization (IVF) procedures remains relatively low, ranging from 4% to 40% depending on the patient's age. It is believed that this is in a large part caused by inaccurate assessment of embryo quality prior to transfer to mothers-to-be. Thus there is a strong need for further refinement of existing selection methods and development of novel, robust and, if only possible, non-invasive procedures to ensure that only embryos with the highest developmental potential are chosen for transfer. In the present review we compare various methods for assessing the quality of preimplantation embryos either currently used in IVF clinics or still to be tested. These methods include assessment of embryonic morphology, the genetic material, the transcriptomes of the oocyte and its accompanying follicular cells, and the embryo's metabolism. We discuss what information these parameters actually provide about the processes occurring in the embryo itself. We also present novel methods for selecting healthy embryos based on most recent advanced time-lapse imaging techniques, which show great promise and are likely to lead to increased in vitro fertilization efficiency., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A longstanding question in mammalian development is whether the divisions that segregate pluripotent progenitor cells for the future embryo from cells that differentiate into extraembryonic structures are asymmetric in cell-fate instructions. The transcription factor Cdx2 plays a key role in the first cell-fate decision. Here, using live-embryo imaging, we show that localization of Cdx2 transcripts becomes asymmetric during development, preceding cell lineage segregation. Cdx2 transcripts preferentially localize apically at the late eight-cell stage and become inherited asymmetrically during divisions that set apart pluripotent and differentiating cells. Asymmetric localization depends on a cis element within the coding region of Cdx2 and requires cell polarization as well as intact microtubule and actin cytoskeletons. Failure to enrich Cdx2 transcripts apically results in a significant decrease in the number of pluripotent cells. We discuss how the asymmetric localization and segregation of Cdx2 transcripts could contribute to multiple mechanisms that establish different cell fates in the mouse embryo., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells of early mouse embryo were considered for a long time to acquire cell fate at random. Recent analyses argue against this simple model., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cell identity is specified in the early mammalian embryo by the generation of precursors for two cell lineages: the pluripotent inner cell mass and differentiating trophectoderm. Here we identify Angiomotin as a key regulator of this process. We show that the loss of Angiomotin, together with Angiomotin-like 2, leads to differentiation of inner cell mass cells and compromised peri-implantation development. We show that Angiomotin regulates localization of Yap, and Yap-binding motifs are required for full activity of Angiomotin. Importantly, we also show that Angiomotin function can compensate for the absence of Lats1/2 kinases, indicating the ability of Angiomotin to bypass the classical Hippo pathway for Yap regulation. In polarized outside cells, Angiomotin localizes apically, pointing to the importance of cell polarity in regulating Yap to promote differentiation. We propose that both Hippo pathway-dependent and Hippo pathway-independent mechanisms regulate Yap localization to set apart pluripotent and differentiated lineages in the pre-implantation mouse embryo.

How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

Plasticity is a well-known feature of mammalian development, and yet very little is known about its underlying mechanism. Here, we establish a model system to examine the extent and limitations of developmental plasticity in living mouse embryos. We show that halved embryos follow the same strict clock of developmental transitions as intact embryos, but their potential is not equal. We have determined that unless a minimum of four pluripotent cells is established before implantation, development will arrest. This failure can be rescued by modulating Fgf and Wnt signaling to enhance pluripotent cell number, allowing the generation of monozygotic twins, which is an otherwise rare phenomenon. Knowledge of the minimum pluripotent-cell number required for development to birth, as well as the different potentials of blastomeres, allowed us to establish a protocol for splitting an embryo into one part that develops to adulthood and another that provides embryonic stem cells for that individual., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone arginine methylation has emerged as an important histone modification involved in gene regulation. Protein arginine methyltransferase (PRMT) 4 and 5 have been shown to play essential roles in early embryonic development and in embryonic stem (ES) cells. Recently, it has been reported that PRMT6-mediated di-methylation of histone H3 at arginine 2 (H3R2me2) can antagonize tri-methylation of histone H3 at lysine 4 (H3K4me3), which marks active genes. However, whether PRMT6 and PRMT6-mediated H3R2me2 play crucial roles in early embryonic development and ES cell identity remain unclear. Here, we have investigated their roles using gain and loss of function studies with mouse ES cells as a model system. We report that Prmt6 and histone H3R2 methylation levels increased when ES cells are induced to differentiate. Consistently, we find that differentiation of ES cells upon upregulation of Prmt6 is associated with decreased expression of pluripotency genes and increased expression of differentiation markers. We also observe that elevation of Prmt6 increases the methylation level of histone H3R2 and decreases H3K4me, Chd1, and Wdr5 levels at the promoter regions of Oct4 and Nanog. Surprisingly, knockdown of Prmt6 also leads to downregulation of pluripotency genes and induction of expression of differentiation markers suggesting that Prmt6 is important for ES cell pluripotency and self-renewal. Our results indicate that a critical level of Prmt6 and histone H3R2me must be maintained in mouse ES cells to sustain their pluripotency.

The development of an anterior-posterior (AP) polarity is a crucial process that in the mouse has been very difficult to analyse, because it takes place as the embryo implants within the mother. To overcome this obstacle, we have established an in-vitro culture system that allows us to follow the step-wise development of anterior visceral endoderm (AVE), critical for establishing AP polarity. Here we use this system to show that the AVE originates in the implanting blastocyst, but that additional cells subsequently acquire AVE characteristics. These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure. Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration. Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

Despite many recent advances in the field of reproductive biology and medicine, the efficiency of in vitro fertilization procedures remains relatively low. There is a need for a reliable and non-invasive method of embryo selection to ensure that only embryos with the highest developmental potential are chosen for transfer to mothers-to-be. Here, we compare various methods currently used for assessing embryonic viability, such as examination of embryonic morphology, quality of the genetic material, or metabolism. Additionally, we discuss novel procedures for embryonic assessment based on advanced time-lapse imaging techniques, which show great promise and may lead to increased in vitro fertilization efficiencies.

Early development of the mouse comprises a sequence of cell fate decisions in which cells are guided along a pathway of restricted potential and increasing specialisation. The first choice faced by cells of the embryo is whether to become trophectoderm (TE) or inner cell mass (ICM); TE is an extra-embryonic tissue which will form the embryonic portion of the placenta, whilst ICM gives rise to cells responsible for generating the foetus. In the second cell fate decision, the ICM is further refined into pluripotent cells forming the future body of the embryo, epiblast (EPI) and extra-embryonic primitive endoderm (PE), a tissue essential for patterning the embryo and establishing the developmental circulation. Understanding this early lineage segregation is critical for informing attempts to capture pluripotency and direct cell fate in vitro. Unlike the predictability of nonmammalian cell fate, development of the mouse embryo retains the flexibility to adapt to changing circumstances during development. Here we describe these first cell fate decisions, how they can be biased whilst maintaining flexibility and, finally, some of the molecular circuitry underlying early fate choice.

Fertilization-induced cytoplasmic flows are a conserved feature of eggs in many species. However, until now the importance of cytoplasmic flows for the development of mammalian embryos has been unknown. Here, by combining a rapid imaging of the freshly fertilized mouse egg with advanced image analysis based on particle image velocimetry, we show that fertilization induces rhythmical cytoplasmic movements that coincide with pulsations of the protrusion forming above the sperm head. We find that these movements are caused by contractions of the actomyosin cytoskeleton triggered by Ca(2+) oscillations induced by fertilization. Most importantly, the relationship between the movements and the events of egg activation makes it possible to use the movements alone to predict developmental potential of the zygote. In conclusion, this method offers, thus far, the earliest and fastest, non-invasive way to predict the viability of eggs fertilized in vitro and therefore can potentially improve greatly the prospects for IVF treatment.

In the mouse embryo, the first differences between cells that result in distinct lineages have long been thought to arise only as a consequence of differential cell positioning at relatively late preimplantation stages. Differences in Oct4 transcription factor kinetics between cells at the 4-8-cell stage are now shown to be predictive of future lineages, providing further evidence for much earlier initiation of cell fate decisions.

The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.

The temporal and spatial segregation of the two extra-embryonic cell lineages, trophectoderm and primitive endoderm (TE and PE respectively), from the pluripotent epiblast (EPI) during mammalian pre-implantation development are prerequisites for the successful implantation of the blastocyst. The mechanisms underlying these earliest stages of development remain a fertile topic for research and informed debate. In recent years novel roles for various transcription factors, polarity factors and signalling cascades have been uncovered. This mini-review seeks to summarise some of this work and to put it into the context of the regulative nature of early mammalian development and to highlight how the increasing evidence of naturally occurring asymmetries and heterogeneity in the embryo can bias specification of the distinct cell types of the blastocyst., (Copyright © 2010 Elsevier Ltd. All rights reserved.)