Nuclear transfer: Progress and quandaries

Cloning mammals by nuclear transfer is a powerful technique that is quickly advancing the development of genetically defined animal models. However, the overall efficiency of nuclear transfer is still very low and several hurdles remain before the power of this technique will be fully harnessed. Among these hurdles include an incomplete understanding of biologic processes that control epigenetic reprogramming of the donor genome following nuclear transfer. Incomplete epigenetic reprogramming is considered the major cause of the developmental failure of cloned embryos and is frequently associated with the disregulation of specific genes. At present, little is known about the developmental mechanism of reconstructed embryos. Therefore, screening strategies to design nuclear transfer protocols that will mimic the epigenetic remodeling occurring in normal embryos and identifying molecular parameters that can assess the developmental potential of pre-implantation embryos are becoming increasingly important. A crucial need at present is to understand the molecular events required for efficient reprogramming of donor genomes after nuclear transfer. This knowledge will help to identify the molecular basis of developmental defects seen in cloned embryos and provide methods for circumventing such problems associated with cloning the future application of this technology

Early alteration of the self-renewal/differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer

Development after nuclear transfer (NT) is subjected to defects originating from both the epiblast and the trophoblast parts of the conceptus and is always accompanied by placentomegaly at term. Here we have investigated the origin of the reprogramming errors affecting the trophoblast lineage in mouse NT embryos. We show that trophoblast stem (TS) cells can be derived from NT embryos (ntTS cells) and used as an experimental in vitro model of trophoblast proliferation and differentiation. Strikingly, TS derivation is more efficient from NT embryos than from controls and ntTS cells exhibit a growth advantage over control TS cells under self-renewal conditions. While epiblast-produced growth factors Fgf4 and Activin exert a fine-tuned control on the balance between self-renewal and differentiation of control TS cells, ntTS cells exhibit a reduced dependency upon their micro-environment. Since the supply of growth factors is known do decrease at the onset of placental formation in vivo we propose that TS cells in NT embryos continue to self-renew during a longer period of time than in fertilized embryo. The resulting increased pool of progenitors could contribute to the enlarged extra-embryonic region observed in the early trophoblast of in vivo grown mouse NT blastocysts that results in placentomegaly

Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency.

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation

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

Background: During 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. Results: Induction 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. Conclusions: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of e

Heterogeneity in Epiblast Stem Cells

Epiblast stem cells (EpiSCs) are pluripotent cells that are derived from mouse embryos at gastrulation stages. They represent the primed state of pluripotency, in which cells are on the verge of differentiation and already express markers of the three primary lineages (mesoderm, endoderm, neurectoderm). EpiSCs display some heterogeneity intra- and inter-cell lines in the expression of some of these lineage markers. We relate this heterogeneity to signalling pathways that are active in EpiSCs, either due to addition of growth factors (FGF2 and activin) in the culture medium, or endogenously active (FGF, Nodal, and Wnt). By modulating Wnt or activin/nodal pathways, cell lines close to EpiSCs but with different properties can be obtained. These signalling pathways are all at work in vivo to pattern the pluripotent epiblast and specify cellular fate