Agnieszka Jedrusik

Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo

Genesis of the trophectoderm and inner cell mass (ICM) lineages occurs in two stages. It is initiated via asymmetric divisions of eight- and 16-cell blastomeres that allocate cells to inner and outer positions, each with different developmental fates. Outside cells become committed to the trophectoderm at the blastocyst stage through Cdx2 activity, but here we show that Cdx2 can also act earlier to influence cell allocation. Increasing Cdx2 levels in individual blastomeres promotes symmetric divisions, thereby allocating more cells to the trophectoderm, whereas reducing Cdx2 promotes asymmetric divisions and consequently contribution to the ICM. Furthermore, both Cdx2 mRNA and protein levels are heterogeneous at the eight-cell stage. This heterogeneity depends on cell origin and has developmental consequences. Cdx2 expression is minimal in cells with unrestricted developmental potential that contribute preferentially to the ICM and is maximal in cells with reduced potential that contribute more to the trophectoderm. Finally, we describe a mutually reinforcing relationship between cellular polarity and Cdx2: Cdx2 influences cell polarity by up-regulating aPKC, but cell polarity also influences Cdx2 through asymmetric distribution of Cdx2 mRNA in polarized blastomeres. Thus, divisions generating inside and outside cells are truly asymmetric with respect to cell fate instructions. These two interacting effects ensure the generation of a stable outer epithelium by the blastocyst stage.

The first cleavage of the mouse zygote predicts the blastocyst axis

One of the unanswered questions in mammalian development is how the embryonic–abembryonic axis of the blastocyst is first established. It is possible that the first cleavage division contributes to this process, because in most mouse embryos the progeny of one two-cell blastomere primarily populate the embryonic part of the blastocyst and the progeny of its sister populate the abembryonic part. However, it is not known whether the embryonic–abembryonic axis is set up by the first cleavage itself, by polarity in the oocyte that then sets the first cleavage plane with respect to the animal pole, or indeed whether it can be divorced entirely from the first cleavage and established in relation to the animal pole. Here we test the importance of the orientation of the first cleavage by imposing an elongated shape on the zygote so that the division no longer passes close to the animal pole, marked by the second polar body. Non-invasive lineage tracing shows that even when the first cleavage occurs along the short axis imposed by this experimental treatment, the progeny of the resulting two-cell blastomeres tend to populate the respective embryonic and abembryonic parts of the blastocyst. Thus, the first cleavage contributes to breaking the symmetry of the embryo, generating blastomeres with different developmental characteristics.

Maternally and zygotically provided Cdx2 have novel and critical roles for early development of the mouse embryo

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.

CARM1 Is Required in Embryonic Stem Cells to Maintain Pluripotency and Resist Differentiation

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 2009;27:2637–2645

The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos

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.

Asymmetric localization of Cdx2 mRNA during the first cell-fate decision in early mouse development.

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.

BMP signalling regulates the pre-implantation development of extra-embryonic cell lineages in the mouse embryo

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 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 down-regulating the pathway using RNAi 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, prior to implantation. Together these findings indicate multiple roles of BMP signalling in the early mouse embryo.

Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition.

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.

Pluripotent state transitions coordinate morphogenesis in mouse and human embryos

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

Oocyte Polarity and Its Developmental Significance

Establishment of the animal-vegetal (AV) axis is one of the most important events of meiotic maturation in mammalian oocytes, as it extensively affects further embryonic development. Initially, in prophase of the first meiotic division (ProI), an oocyte is radially symmetric, with a nucleus localized in the cell centre. After resumption of meiosis, metaphase I (MetI) spindle is moved from the central position towards the cortex, marking an animal pole of the oocyte. Translocation of the meiotic spindle depends on actin cytoskeleton and leads to an extensive reorganization of the animal cortex, an event regulated by complex molecular pathways. Asymmetric localization of the oocyte chromatin is maintained in the metaphase II (Met II) stage. Migration of the spindle to the cortex ensures that both meiotic divisions occur in an asymmetric manner giving rise to small polar bodies and the big egg cell containing most of the maternal factors stored during oogenesis. Moreover, cortical reorganization caused by translocation of the oocyte chromatin prevents egg-sperm fusion in the vicinity of the animal pole and in consequence precocious mixing of maternal and paternal chromosomes that could disturb proper segregation of genetic material during the second meiotic division. Finally, recent research provides evidence that the AV axis formed in the oocyte may influence embryonic fate of the blastomeres, as cells containing either animal or vegetal components are differentially predisposed. We would like to present here the current stage of knowledge regarding molecular mechanism of AV axis formation in mammalian oocytes and developmental significance of this process.