Magdalena Zernicka-goetz | University of Cambridge (original) (raw)

Papers by Magdalena Zernicka-goetz

Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating... more 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. STEM CELLS ; available online without subscription thorugh the open access option. V C AlphaMed

Developmental Biology, 2014

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the tr... more 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 mostbut not allcells 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.

Developmental Biology, 2015

The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which give... more 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.

Nature Communications, 2012

The development of an anterior -posterior (AP) polarity is a crucial process that in the mouse ha... more The development of an anterior -posterior (AP) polarity is a crucial process that in the mouse has been very diffi cult 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.

Developmental Biology, 2007

Initiation of the development of the anterior–posterior axis in the mouse embryo has been thought... more 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

Mechanisms of Development, 2006

The development of the anterior–posterior (AP) axis in the mammalian embryo is controlled by inte... more The development of the anterior–posterior (AP) axis in the mammalian embryo is controlled by interactions between embryonic and extraembryonic tissues. It is well established that one of these extraembryonic tissues, the anterior visceral endoderm (AVE), can repress posterior cell fate and that signalling from the other, the extraembryonic ectoderm (ExE), is required for posterior patterning. Here, we show that signals

Nature Communications, 2014

Pre-implantation development requires the specification and organization of embryonic and extra-e... more 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.

International Journal of Developmental Biology, 2006

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA meth... more Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. Here we have examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. We show ,that H3.3 is

BMC developmental biology, 2005

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombi... more 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 ...

Nature, 2006

Hiiragi et al. compare our model of the developing mouse egg with theirs. They seem to present pa... more Hiiragi et al. compare our model of the developing mouse egg with theirs. They seem to present patterning as equivalent to determination, but this is confusing as patterning does not have to mean determination. We have never stated that mouse embryo development is determined. Mouse development is regulative rather than determinative, and this can be explained in two ways: first,

Proceedings of the National Academy of Sciences, 2010

A crucial question in mammalian development is how cells of the early embryo differentiate into d... more A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. The first decision is taken when cells undertake waves of asymmetric division that generate one daughter on the inside and one on the outside of the embryo. After this division, some cells on the inside remain pluripotent and give rise to the epiblast, and hence the future body, whereas others develop into the primitive endoderm, an extraembryonic tissue. How the fate of these inside cells is decided is unknown: Is the process random, or is it related to their developmental origins? To address this question, we traced all cells by livecell imaging in intact, unmanipulated embryos until the epiblast and primitive endoderm became distinct. This analysis revealed that inner cell mass (ICM) cells have unrestricted developmental potential. However, cells internalized by the first wave of asymmetric divisions are biased toward forming pluripotent epiblast, whereas cells internalized in the next two waves of divisions are strongly biased toward forming primitive endoderm. Moreover, we show that cells internalized by the second wave up-regulate expression of Gata6 and Sox17, and changing the expression of these genes determines whether the cells become primitive endoderm. Finally, with our ability to determine the origin of cells, we find that inside cells that are mispositioned when they are born can sort into the correct layer. In conclusion, we propose a model in which the timing of cell internalization, cell position, and cell sorting combine to determine distinct lineages of the preimplantation mouse embryo.

Nature Cell Biology, 2002

The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals su... more The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals suggests that even in highly regulative embryos such as the mouse, division patterns might be important for development 1-4 . Indeed, the first cleavage divides the fertilized mouse egg into two cells: one cell that contributes predominantly to the embryonic part of the blastocyst, and one that contributes to the abembryonic part 5,6 . Here we show, by removing, transplanting or duplicating the animal or vegetal poles of the mouse egg, that a spatial cue at the animal pole orients the plane of this initial division. Embryos with duplicated animal, but not vegetal, poles show abnormalities in chromosome segregation that compromise their development. Our results show that localized factors in the mammalian egg orient the spindle and so define the initial cleavage plane. In increased dosage, however, these factors are detrimental to the correct execution of division.

Nature, 2007

It has been generally accepted that the mammalian embryo starts its development with all cells id... more 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 as early as the four-cell stage 1-4 . These differences depend on the orientation and order of the cleavage divisions that generated them 2,5 . Because epigenetic marks are suggested to be involved in sustaining pluripotency 6,7 , we considered that such developmental properties might be achieved through epigenetic mechanisms. Here we show that modification of histone H3, through the methylation of specific arginine residues, is correlated with cell fate and potency. Levels of H3 methylation at specific arginine residues are maximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. 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 that 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.

Molecular Biology of the Cell, 2008

Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction... more 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.

Journal of Cell Science, 2005

Fertility and Sterility, 2012

Objective: To evaluate the imaging of cytoplasmic movements in human oocytes as a potential metho... more Objective: To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca 2þ 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 (z) cRNA and a Ca 2þ -sensitive fluorescent dye. Main Outcome Measure(s): Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca 2þ oscillations using a Ca 2þ -sensitive fluorescent dye. Result(s): Microinjection of PLCz cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca 2þ oscillations. Each transient Ca 2þ 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 Ca 2þ 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 Ca 2þ oscillations in human zygotes. (Fertil Steril Ò 2012;97:742-7.

Differentiation, 2003

Embryonic germ cells (EGCs) derived from mouse primordial germ cells (PGCs) are known both to col... more Embryonic germ cells (EGCs) derived from mouse primordial germ cells (PGCs) are known both to colonize all cell lineages of the fetus and to make tumors in vivo. When aggregated with eight-cell embryos, EGCs from a new EGC line expressing green fluorescent protein (GFP) were found to contribute preferentially to the epiblast but unexpectedly were also capable of colonizing primary endoderm. When injected under the kidney capsule, EGCs derived from 12.5 days post coitum (dpc) PGCs formed differentiated tumors. The ability of EGCs to differentiate in an organ culture system depends upon their partners in cell culture. When EGCs, marked with a LacZ transgene, were mixed with disaggregated and reaggregated mouse fetal lung in an organ culture system, they remained undifferentiated. In urogenital ridge reaggregates on the other hand, some EGCs were capable of differentiating to form small epithelial cysts.