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1.  Spindle Formation in the Mouse Embryo Requires Plk4 in the Absence of Centrioles 
Developmental Cell  2013;27(5):586-597.
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.
•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.
PMCID: PMC3898710  PMID: 24268700
2.  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.
PMCID: PMC3843820  PMID: 24258274
mouse embryo; cell lineage; heterogeneity; Fgf signalling; bias
3.  Asymmetric Localization of Cdx2 mRNA during the First Cell-Fate Decision in Early Mouse Development 
Cell Reports  2013;3(2):442-457.
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
► 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.
PMCID: PMC3607255  PMID: 23375373
4.  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.
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.
PMCID: PMC3585521  PMID: 23077180
Cell commitment; Epigenetic stability; Induced pluripotency; macroH2A; Nuclear reprogramming
5.  Developmental Plasticity Is Bound by Pluripotency and the Fgf and Wnt Signaling Pathways 
Cell Reports  2012;2(4):756-765.
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
► 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.
PMCID: PMC3607220  PMID: 23041313
6.  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.
PMCID: PMC3369238  PMID: 22685489
7.  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.
PMCID: PMC3353120  PMID: 17215844
8.  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.
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.
PMCID: PMC3353121  PMID: 17662710
9.  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.
PMCID: PMC3353122  PMID: 17005174
Dishevelled; mouse; blastocyst; preimplantation; development
10.  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.
PMCID: PMC3353123  PMID: 19422818
mouse blastocyst; cell lineage; cell movement; Gata6; Wnt; inner cell mass; primitive endoderm
11.  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.
PMCID: PMC3342679  PMID: 18657169
anterior visceral endoderm; anterior-posterior axis; Bmp4; Cerberus-like; electroporation; mouse embryo; RNA interference
12.  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.
To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca2+ oscillations during activation.
Test of a laboratory technique.
University medical school research laboratory.
Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles.
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.
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.
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.
PMCID: PMC3334266  PMID: 22217962
Oocyte; zygote; calcium; movement; phospholipase C zeta; cross correlation; particle image velocimetry
13.  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.
PMCID: PMC3265380  PMID: 21829179
14.  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.
PMCID: PMC2912351  PMID: 20554762
15.  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.
PMCID: PMC2954319  PMID: 20430022
Cdx2; Trophectoderm; Mouse embryo; Polarisation; Cell death; Compaction
16.  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.
PMCID: PMC2655627  PMID: 18234722
17.  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.
PMCID: PMC2555945  PMID: 18701707
18.  Regionalisation of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder 
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.
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.
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.
PMCID: PMC1978209  PMID: 17705827
19.  Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation 
To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region.
To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function.
Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation.
PMCID: PMC1821012  PMID: 17302988
20.  Role of TIF1α as a modulator of embryonic transcription in the mouse zygote 
The Journal of Cell Biology  2006;174(3):329-338.
The first events of the development of any embryo are under maternal control until the zygotic genome becomes activated. In the mouse embryo, the major wave of transcription activation occurs at the 2-cell stage, but transcription starts already at the zygote (1-cell) stage. Very little is known about the molecules involved in this process. We show that the transcription intermediary factor 1 α (TIF1α) is involved in modulating gene expression during the first wave of transcription activation. At the onset of genome activation, TIF1α translocates from the cytoplasm into the pronuclei to sites of active transcription. These sites are enriched with the chromatin remodelers BRG-1 and SNF2H. When we ablate TIF1α through either RNA interference (RNAi) or microinjection of specific antibodies into zygotes, most of the embryos arrest their development at the 2–4-cell stage transition. The ablation of TIF1α leads to mislocalization of RNA polymerase II and the chromatin remodelers SNF2H and BRG-1. Using a chromatin immunoprecipitation cloning approach, we identify genes that are regulated by TIF1α in the zygote and find that transcription of these genes is misregulated upon TIF1α ablation. We further show that the expression of some of these genes is dependent on SNF2H and that RNAi for SNF2H compromises development, suggesting that TIF1α mediates activation of gene expression in the zygote via SNF2H. These studies indicate that TIF1α is a factor that modulates the expression of a set of genes during the first wave of genome activation in the mouse embryo.
PMCID: PMC2064229  PMID: 16880268
21.  Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi 
Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.
We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.
Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.
PMCID: PMC1363358  PMID: 16381610
22.  Dynamics of anterior–posterior axis formation in the developing mouse embryo 
Nature Communications  2012;3:673-.
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.
Detailed analysis of axis development in mouse embryo has been limited. Morris et al. developed an in vitro culture technique that enables the real-time observation of an anterior visceral endoderm formation and show that cell marker asymmetry within the AVE subdomain dictates the direction of the AVE migration.
PMCID: PMC3293425  PMID: 22334076
23.  Angiomotin prevents pluripotent lineage differentiation in mouse embryos via Hippo pathway-dependent and -independent mechanisms 
Nature Communications  2013;4:2251.
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.
Angiomotins retain the transcription co-activator YAP in the cytoplasm and thereby regulate the Hippo pathway in mammalian cultured cells. Here Leung and Zernicka-Goetz show that Angiomotin family members prevent the differentiation of inner cell mass cells in the mouse blastocyst, via both Hippo pathway-dependent and -independent mechanisms.
PMCID: PMC3741640  PMID: 23903990

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