Alice Jouneau

Developmental Potential of Mouse Embryos Reconstructed from Metaphase Embryonic Stem Cell Nuclei

Abstract Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogenous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56–70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.

Developmental abnormalities of NT mouse embryos appear early after implantation.

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.

Expression of Pluripotency Master Regulators during Two Key Developmental Transitions: EGA and Early Lineage Specification in the Bovine Embryo

Pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. In bovine, however, little information is available about dynamics of pluripotency genes during these processes. In this study, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, having similar in vitro but different full term development rates. Our findings affirm: firstly, the core triad of pluripotency genes is probably not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Secondly, an earlier ICM specification of transcripts and proteins of SOX2 and NANOG makes them pertinent candidates of bovine pluripotent lineage specification than OCT4. Thirdly, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development.

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles.

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming.

Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis.

Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the cloning technique which has not yet been well investigated. There are two main procedures for transferring nuclei into enucleated oocytes: fusion and piezoelectric microinjection, the latter being used mostly in mice. We have, therefore, decided to compare the quality and the developmental ability, both in vivo and in vitro, of embryos reconstructed with electrofusion or piezoelectric injection. In addition, the effect of piezo setups of differing electric strengths was investigated. Along with the record of the rate of development, we compared the nuclear integrity in the blastomeres during the first cleavages as well as the morphological and cellular quality of the blastocysts. Our results show that the piezo-assisted micromanipulation can induce DNA damage in the reconstructed embryos, apoptosis, and reduced cell numbers in blastocysts as well as a lower rate of development to term. Even if piezo-driven injection facilitates a faster and more efficient rate of reconstruction, it should be used with precaution and with as low parameters as possible.

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

BackgroundDuring early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq.ResultsInduction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs.ConclusionsThe gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors

Transfer of somatic cell nuclei to enucleated eggs and ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, these methods are poorly efficient, and other unknown factors might be required to increase their success rate. Here we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development, and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4, and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M-phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies, and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M-phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergizes with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial, because none of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei.

Efficient derivation of bovine embryonic stem cells needs more than active core pluripotency factors

Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self‐renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day‐8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC‐like and EpiSC‐like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long‐term pluripotency ex vivo, as was reported for mouse and pig EpiSCs. Mol. Reprod. Dev. 79:461–477, 2012.

Embryonic Stem Cells in Cattle

Because of the potential use of embryonic stem cells (ESC), especially for genetic modifications, there is great interest in establishing domestic animals-related ESCs. Unfortunately, despite considerable efforts, validated ESC lines in species other than mice and primates are yet to be isolated. In this paper, we will summarize the current knowledge on bovine ESCs in an attempt to understand why derivation of domestic animal ESC is still unsuccessful and we will discuss some promising future approaches.