The chromatin modifier Satb1 regulates cell fate through Fgf signalling in the early mouse embryo

The separation of embryonic from extra-embryonic tissues within the inner cell mass to generate the epiblast (EPI), which will form the new organism, from the primitive endoderm (PE), which will form the yolk sac, is a crucial developmental decision. Here, we identify a chromatin modifier, Satb1, with a distinct role in this decision. Satb1 is differentially expressed within 16-cell-stage embryos, with higher expression levels in the inner cell mass progenitor cells. Depleting Satb1 increases the number of EPI cells at the expense of PE. This phenotype can be rescued by simultaneous depletion of both Satb1 and Satb2, owing to their antagonistic effect on the pluripotency regulator Nanog. Consequently, increasing Satb1 expression leads to differentiation into PE and a decrease in EPI, as a result of the modulation of expression of several pluripotency- and differentiation-related genes by Satb1. Finally, we show that Satb1 is a downstream target of the Fgf signalling pathway, linking chromatin modification and Fgf signalling. Together, these results identify a role for Satb1 in the lineage choice between pluripotency and differentiation and further our understanding of early embryonic lineage segregation.We are grateful to the Wellcome Trust Senior Research Fellowship to M.Z.-G. who funded this work (grant 098287)

Expression of SARS-CoV-2 receptor ACE2 and the protease TMPRSS2 suggests susceptibility of the human embryo in the first trimester.

While initially recognized as causing respiratory disease, the SARS-CoV-2 virus also affects many other organs leading to other complications. It has emerged that advanced age and obesity are risk factors for complications but questions concerning the potential effects on fetal health and successful pregnancy for those infected with SARS-CoV-2 remain largely unanswered. Here, we examine human pre-gastrulation embryos to determine the expression patterns of the genes ACE2, encoding the SARS-CoV-2 receptor, and TMPRSS2, encoding a protease that cleaves both the viral spike protein and the ACE2 receptor to facilitate infection. We show expression and co-expression of these genes in the trophoblast of the blastocyst and syncytiotrophoblast and hypoblast of the implantation stages, which develop into tissues that interact with the maternal blood supply for nutrient exchange. Expression of ACE2 and TMPRSS2 in these tissues raises the possibility for vertical transmission and indicates that further work is required to understand potential risks to implantation, placental health and fetal health that require further study

RNA Interference by Production of Short Hairpin dsRNA in ES Cells, Their Differentiated Derivatives, and in Somatic Cell Lines

dsRNA of several hundred nucleotides in length is effective at interfering with gene expression in mouse oocytes, pre-implantation embryos, and embryonic stem (ES) cells but is not as efficient in differentiated cell lines. Here we describe a method to achieve RNA interference in totipotent and differentiated ES cells together with a wide range of other mammalian cell types that is both simple and efficient. It utilizes a linearized plasmid that directs the expression of a hairpin RNA with a 22-nucleotide-paired region. This molecule has a 13-nucleotide 5′ overhang that would be subject to capping on its 5′ phosphoryl group and thus differs from the ideal structure suggested for effective small interfering RNAs. Thus, it appears either that the structure of small inhibitory RNA molecules may not need to be as precise as previously thought or that such a transcript is efficiently processed to a form that is effective in interfering with gene expression

Integrin beta 1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

At implantation, the embryo establishes contacts with the maternal endometrium. This stage is associated with a high incidence of preclinical pregnancy losses. While the maternal factors underlying uterine receptivity have been investigated, the signals required by the embryo for successful peri-implantation development remain elusive. To explore these, we studied integrin beta 1 signaling, as embryos deficient for this receptor degenerate at implantation. We demonstrate that the coordinated action of pro-survival signals and localized actomyosin suppression via integrin beta 1 permits the development of the embryo beyond implantation. Failure of either process leads to developmental arrest and apoptosis. Pharmacological stimulation through fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1), coupled with ROCK-mediated actomyosin inhibition, rescues the deficiency of integrin beta 1, promoting progression to post-implantation stages. Mutual exclusion between integrin beta 1 and actomyosin seems to be conserved in the human embryo, suggesting the possibility that these mechanisms could also underlie the transition of the human epiblast from pre- to post-implantation

Actomyosin polarisation through PLC-PKC triggers symmetry breaking of the mouse embryo

Establishment of cell polarity in the mammalian embryo is fundamental for the first cell fate decision that sets aside progenitor cells for both the new organism and the placenta. Yet the sequence of events and molecular mechanism that trigger this process remain unknown. Here, we show that de novo polarisation of the mouse embryo occurs in two distinct phases at the 8-cell stage. In the first phase, an apical actomyosin network is formed. This is a pre-requisite for the second phase, in which the Par complex localises to the apical domain, excluding actomyosin and forming a mature apical cap. Using a variety of approaches, we also show that phospholipase C-mediated PIP2 hydrolysis is necessary and sufficient to trigger the polarisation of actomyosin through the Rho-mediated recruitment of myosin II to the apical cortex. Together, these results reveal the molecular framework that triggers de novo polarisation of the mouse embryo.This work was supported by the Wellcome Trust. M.Z.G. is a Wellcome Trust Senior Research Fellow, M.Z. is supported by the Cambridge Trust, and M.N.S. is an EMBO Postdoctoral Fellow

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.We thank the Wellcome Trust (Grant no. 098287/Z/12/Z) for supporting this work.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0012160614006307#

Delayed APC/C activation extends the first mitosis of mouse embryos

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

Regionalization 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.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

PAR6C polarity protein expression pattern in male and female bovine embryos.

Proceedings of the 28th Annual Meeting of the Brazilian Embryo Technology Society (SBTE), August 14 to 17th, 2014, Natal, RN, Brazil. Abstracts

An indelible lineage marker for Xenopus using a mutated green fluorescent protein

We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed