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1.  Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition 
Developmental Biology  2015;398(2):147-152.
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.
doi:10.1016/j.ydbio.2014.12.004
PMCID: PMC4319684  PMID: 25512302
Mouse embryo; Cdx2; Maternal-zygotic knockout; Trophectoderm
4.  Developmental plasticity, cell fate specification and morphogenesis 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.
doi:10.1098/rstb.2013.0538
PMCID: PMC4216461  PMID: 25349447
embryo; morphogenesis; cell fate; differentiation; pluripotency
5.  CARM1 is Required in Embryonic Stem Cells to Maintain Pluripotency and Resist Differentiation 
Stem cells (Dayton, Ohio)  2009;27(11):2637-2645.
Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating pluripotency in mouse embryos. Blastomeres of four-cell embryos with high H3 methylation at these sites show unrestricted potential, whereas those with lower levels cannot support development when aggregated in chimeras of like cells. Increasing histone H3 methylation, through expression of coactivator-associated-protein-arginine-methyltransferase 1 (CARM1) in embryos, elevates expression of key pluripotency genes and directs cells to the pluripotent inner cell mass. We demonstrate CARM1 is also required for the self-renewal and pluripotency of embryonic stem (ES) cells. In ES cells, CARM1 depletion downregulates pluripotency genes leading to their differentiation. CARM1 associates with Oct4/Pou5f1 and Sox2 promoters that display detectable levels of R17/26 histone H3 methylation. In CARM1 overexpressing ES cells, histone H3 arginine methylation is also at the Nanog promoter to which CARM1 now associates. Such cells express Nanog at elevated levels and delay their response to differentiation signals. Thus, like in four-cell embryo blastomeres, histone H3 arginine methylation by CARM1 in ES cells allows epigenetic modulation of pluripotency.
doi:10.1002/stem.131
PMCID: PMC4135545  PMID: 19544422
CARM1; ES cells; pluripotency; differentiation; chromatin; arginine histone methylation
6.  The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo 
Developmental Biology  2014;392(2):133-140.
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.
Highlights
•In 8-cell embryo most of the nuclei move from apical to basal positions.•The nuclear movement depends on microtubules and kinesins.•Only blastomeres with basally located nuclei can divide asymmetrically.•Blastomeres with apically located nuclei divide only symmetrically.•Position of the nucleus is regulated by interdependence of Cdx2 and cell polarity
doi:10.1016/j.ydbio.2014.05.009
PMCID: PMC4111899  PMID: 24855000
Mammalian embryo; Preimplantation development; Division plane; Cell polarity; Cytoskeleton
7.  Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon Implantation 
Cell  2014;156(5):1032-1044.
Summary
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.
Graphical Abstract
Highlights
•Apoptosis is not essential for the peri-implantation morphogenesis, as believed•Basal membrane proteins create a niche for EPI and drive morphogenesis in ES cells•Polarization and apical constriction reorganize the EPI into a rosette•The proamniotic cavity is formed through hollowing mechanism
The first morphogenetic event by pluripotent stem cells of mouse blastocyst entails epiblast cell polarization and arrangement into a rosette pattern, which parts at its center, leading to cavity formation.
doi:10.1016/j.cell.2014.01.023
PMCID: PMC3991392  PMID: 24529478
8.  Spindle Formation in the Mouse Embryo Requires Plk4 in the Absence of Centrioles 
Developmental Cell  2013;27(5):586-597.
Summary
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.
Highlights
•Plk4 is at acentriolar MTOCs and spindle poles in mouse embryos•Plk4 is essential for acentriolar spindle assembly•Depletion of maternal Plk4 prevents normal nucleation and growth of microtubules•Plk4 MT-nucleating function depends on its kinase activity and its partner, Cep152
During the first five rounds of division in the mouse embryo, spindles assemble in the absence of centrioles. Here, Coelho et al. show that protein kinase Plk4, the master regulator of centriole formation, and its partner protein Cep152 are also essential for assembly of acentriolar spindles in the mouse embryo.
doi:10.1016/j.devcel.2013.09.029
PMCID: PMC3898710  PMID: 24268700
9.  The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos 
Open Biology  2013;3(11):130104.
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.
doi:10.1098/rsob.130104
PMCID: PMC3843820  PMID: 24258274
mouse embryo; cell lineage; heterogeneity; Fgf signalling; bias
10.  Asymmetric Localization of Cdx2 mRNA during the First Cell-Fate Decision in Early Mouse Development 
Cell Reports  2013;3(2):442-457.
Summary
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.
Graphical Abstract
Highlights
► Cdx2 mRNA localizes apically upon embryo compaction at the eight-cell stage ► Cdx2 mRNA is inherited asymmetrically during asymmetric divisions ► Localization requires cell polarization and intact cytoskeletal components ► Mislocalization of Cdx2 mRNA decreases the number of pluripotent cells
A longstanding question in mouse development is whether divisions that set apart pluripotent and differentiating cells are asymmetric in cell-fate instructions. Cdx2 is a key factor in this first fate decision. Zernicka-Goetz and colleagues use live embryo imaging to show that localization and inheritance of Cdx2 transcripts become asymmetric as pluripotent and differentiating lineages segregate. They demonstrate that asymmetric transcript localization requires cell polarization as well as intact microtubule and actin cytoskeletons, and that mislocalization of Cdx2 transcripts significantly decreases the number of pluripotent cells.
doi:10.1016/j.celrep.2013.01.006
PMCID: PMC3607255  PMID: 23375373
11.  Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency 
Journal of Cell Science  2012;125(24):6094-6104.
Summary
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.
doi:10.1242/jcs.113019
PMCID: PMC3585521  PMID: 23077180
Cell commitment; Epigenetic stability; Induced pluripotency; macroH2A; Nuclear reprogramming
12.  Developmental Plasticity Is Bound by Pluripotency and the Fgf and Wnt Signaling Pathways 
Cell Reports  2012;2(4):756-765.
Summary
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.
Graphical Abstract
Highlights
► Half embryos follow the same clock as intact embryos, but their potential is not equal ► To support development, four pluripotent cells must be generated before implantation ► Fgf/Wnt signal modulation enhances pluripotency to rescue half-embryo development ► ESCs and a viable mouse can be derived from a single embryo with high efficiency
It is a well-known feature of early mouse development that healthy mice can form from embryos split into two parts. How the mouse achieves this remarkable feat is unknown; thus, Morris, Guo, and Zernicka-Goetz examined this phenomenon by filming halved embryos as they developed. They found that some half embryos do not make enough pluripotent cells to support development, but this deficiency can be rescued by modulating Fgf and Wnt signaling. Using this rescue, the authors were able to derive embryonic stem cells without compromising embryo viability.
doi:10.1016/j.celrep.2012.08.029
PMCID: PMC3607220  PMID: 23041313
13.  Advances in embryo selection methods 
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.
doi:10.3410/B4-11
PMCID: PMC3369238  PMID: 22685489
14.  Histone arginine methylation regulates cell fate and pluripotency in the early mouse embryo 
Nature  2007;445(7124):214-218.
It has been generally accepted that the mammalian embryo starts its development with all cells identical and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency already by the 4-cell-stage1-4. Such differences depend on the orientation and order of the cleavage divisions that generated them2,5. Since epigenetic marks are suggested to be involved in sustaining pluripotency6,7, we considered that such developmental properties might be achieved through epigenetic mechanisms. Here, we show that modification of histone H3, through methylation of specific arginine residues, correlates with cell fate and potency. Levels of H3 methylation at specific arginines are maximal in 4-cell blastomeres that will contribute to the ICM and polar trophectoderm and undertake full development when combined together in chimeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. We confirm this prediction by overexpressing the H3-specific arginine methyltransferase, CARM1, in individual blastomeres and show this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, our results identify specific histone modifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination.
doi:10.1038/nature05458
PMCID: PMC3353120  PMID: 17215844
15.  The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation 
Developmental biology  2007;309(1):97-112.
Summary
Initiation of the development of the anterior-posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, pre-implantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early peri-implantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, while it is reinforced in the distal-lateral domain. Our time-lapse studies demonstrate that this distal-lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior-posterior axis is already anticipated before AVE migration.
doi:10.1016/j.ydbio.2007.06.020
PMCID: PMC3353121  PMID: 17662710
16.  Dishevelled Proteins Regulate Cell Adhesion in Mouse Blastocyst and Serve to Monitor Changes in Wnt Signaling 
Developmental biology  2006;302(1):40-49.
Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of β-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell-cell adhesion through the non-canonical Wnt pathway in blastocysts.
doi:10.1016/j.ydbio.2006.08.036
PMCID: PMC3353122  PMID: 17005174
Dishevelled; mouse; blastocyst; preimplantation; development
17.  Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst 
Developmental biology  2009;331(2):210-221.
In the mouse blastocyst, some cells of the inner cell mass (ICM) develop into primitive endoderm (PE) at the surface, while deeper cells form the epiblast. It remained unclear whether the position of cells determines their fate, such that gene expression is adjusted to cell position, or if cells are pre-specified at random positions and then sort. We have tracked and characterised dynamics of all ICM cells from the early to late blastocyst stage. Time-lapse microscopy in H2B-EGFP embryos shows that a large proportion of ICM cells change position between the surface and deeper compartments. Most of this cell movement depends on actin and is associated with cell protrusions. We also find that while most cells are precursors for only one lineage, some give rise to both, indicating that lineage segregation is not complete in the early ICM. Finally, changing the expression levels of the PE marker Gata6 reveals that it is required in surface cells but not sufficient for the re-positioning of deeper cells. We provide evidence that Wnt9A, known to be expressed in the surface ICM, facilitates re-positioning of Gata6-expressing cells. Combining these experimental results with computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.
doi:10.1016/j.ydbio.2009.04.036
PMCID: PMC3353123  PMID: 19422818
mouse blastocyst; cell lineage; cell movement; Gata6; Wnt; inner cell mass; primitive endoderm
18.  Bone morphogenetic protein 4 signaling regulates development of the anterior visceral endoderm in the mouse embryo 
The extraembryonic ectoderm (ExE) of the mouse conceptus is known to play a role in embryo patterning by signaling to the underlying epiblast and surrounding visceral endoderm. Bmp4 is one of the key ExE signaling molecules and has been recently implicated to participate in regulating development and migration of the anterior visceral endoderm (AVE). However, it remains unclear when exactly BMP4 signaling starts to regulate AVE positioning. To examine this, we have chosen to affect BMP4 function at two different time points, at embryonic day 5.25 (E5.25), thus before AVE migration, and E5.75, just after AVE migration. To this end, an RNAi technique was used, which consisted of the injection of Bmp4 dsRNA into the proamniotic cavity of the egg cylinder followed by its targeted electroporation into the ExE. This resulted in specific knockdown of Bmp4. It was found that Bmp4 RNAi at E5.25, but not at E5.75, led to an abnormal pattern of expression of the AVE marker Cerberus-like. Thus, BMP4 signaling appears to affect the expression of Cer1 at a specific time window. This RNAi approach provides a convenient means to study spatial and temporal function of genes shortly after embryo implantation.
doi:10.1111/j.1440-169X.2008.01059.x
PMCID: PMC3342679  PMID: 18657169
anterior visceral endoderm; anterior-posterior axis; Bmp4; Cerberus-like; electroporation; mouse embryo; RNA interference
19.  Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection 
Fertility and Sterility  2012;97(3):742-747.
Objective
To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca2+ oscillations during activation.
Design
Test of a laboratory technique.
Setting
University medical school research laboratory.
Patient(s)
Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles.
Intervention(s)
Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca2+-sensitive fluorescent dye.
Main Outcome Measure(s)
Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca2+ oscillations using a Ca2+-sensitive fluorescent dye.
Result(s)
Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca2+ oscillations. Each transient Ca2+ concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis.
Conclusion(s)
The occurrence and frequency of cytoplasmic Ca2+ oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca2+ oscillations in human zygotes.
doi:10.1016/j.fertnstert.2011.12.013
PMCID: PMC3334266  PMID: 22217962
Oocyte; zygote; calcium; movement; phospholipase C zeta; cross correlation; particle image velocimetry
20.  Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability 
Nature Communications  2011;2:417-.
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 Ca2+ 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.
Cytoplasmic flows—the movement of cytoplasmic material—can be detected following the fertilization of an egg by a sperm in many species. In this study, rhythmic cytoplasmic flows are shown to be induced in mice by calcium-induced cytoskeleton contractions which could be used to predict the successful outcome of fertilization.
doi:10.1038/ncomms1424
PMCID: PMC3265380  PMID: 21829179
21.  Epigenetic Modification Affecting Expression of Cell Polarity and Cell Fate Genes to Regulate Lineage Specification in the Early Mouse Embryo 
Molecular Biology of the Cell  2010;21(15):2649-2660.
In this study, cell behavior and blastomere polarity are examined in the context of Carm1 overexpression in the preimplantation mouse embryo. The results suggest that Carm1 levels can affect the expression of key cell polarity and fate-determining in association with changes in cell behavior and lineage allocation.
Formation of inner and outer cells of the mouse embryo distinguishes pluripotent inner cell mass (ICM) from differentiating trophectoderm (TE). Carm1, which methylates histone H3R17 and R26, directs cells to ICM rather that TE. To understand the mechanism by which this epigenetic modification directs cell fate, we generated embryos with in vivo–labeled cells of different Carm1 levels, using time-lapse imaging to reveal dynamics of their behavior, and related this to cell polarization. This shows that Carm1 affects cell fate by promoting asymmetric divisions, that direct one daughter cell inside, and cell engulfment, where neighboring cells with lower Carm1 levels compete for outside positions. This is associated with changes to the expression pattern and spatial distribution of cell polarity proteins: Cells with higher Carm1 levels show reduced expression and apical localization of Par3 and a dramatic increase in expression of PKCII, antagonist of the apical protein aPKC. Expression and basolateral localization of the mouse Par1 homologue, EMK1, increases concomitantly. Increased Carm1 also reduces Cdx2 expression, a transcription factor key for TE differentiation. These results demonstrate how the extent of a specific epigenetic modification could affect expression of cell polarity and fate-determining genes to ensure lineage allocation in the mouse embryo.
doi:10.1091/mbc.E10-01-0053
PMCID: PMC2912351  PMID: 20554762
22.  Maternally and zygotically provided Cdx2 have novel and critical roles for early development of the mouse embryo 
Developmental Biology  2010;344(1-2):66-78.
Divisions of polarised blastomeres that allocate polar cells to outer and apolar cells to inner positions initiate the first cell fate decision in the mouse embryo. Subsequently, outer cells differentiate into trophectoderm while inner cells retain pluripotency to become inner cell mass (ICM) of the blastocyst. Elimination of zygotic expression of trophectoderm-specific transcription factor Cdx2 leads to defects in the maintenance of the blastocyst cavity, suggesting that it participates only in the late stage of trophectoderm formation. However, we now find that mouse embryos also have a maternally provided pool of Cdx2 mRNA. Moreover, depletion of both maternal and zygotic Cdx2 from immediately after fertilization by three independent approaches, dsRNAi, siRNAi and morpholino oligonucleotides, leads to developmental arrest at much earlier stages than expected from elimination of only zygotic Cdx2. This developmental arrest is associated with defects in cell polarisation, reflected by expression and localisation of cell polarity molecules such as Par3 and aPKC and cell compaction at the 8- and 16-cell stages. Cells deprived of Cdx2 show delayed development with increased cell cycle length, irregular cell division and increased incidence of apoptosis. Although some Cdx2-depleted embryos initiate cavitation, the cavity cannot be maintained. Furthermore, expression of trophectoderm-specific genes, Gata3 and Eomes, and also the trophectoderm-specific cytokeratin intermediate filament, recognised by Troma1, are greatly reduced or undetectable. Taken together, our results indicate that Cdx2 participates in two steps leading to trophectoderm specification: appropriate polarisation of blastomeres at the 8- and 16-cell stage and then the maintenance of trophectoderm lineage-specific differentiation.
doi:10.1016/j.ydbio.2010.04.017
PMCID: PMC2954319  PMID: 20430022
Cdx2; Trophectoderm; Mouse embryo; Polarisation; Cell death; Compaction
23.  Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions 
Development (Cambridge, England)  2008;135(5):953-962.
Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern; whether it is lineage-dependent or occurs at random; or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and so embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together our results suggest that two steps influence allocation of cells to the blastocyst. The first step involving orientation of 2- to 4-cell divisions along the animal-vegetal axis can affect the second step, the establishment of inside and outside cell populations by asymmetric 8-32-cell divisions.
doi:10.1242/dev.014316
PMCID: PMC2655627  PMID: 18234722
24.  Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition 
Molecular Biology of the Cell  2008;19(10):4383-4392.
Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA “slicer” activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery.
doi:10.1091/mbc.E08-02-0219
PMCID: PMC2555945  PMID: 18701707
25.  Regionalisation of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder 
Background
Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue.
Results
As a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5.
Conclusion
We propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.
doi:10.1186/1471-213X-7-96
PMCID: PMC1978209  PMID: 17705827

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