RIF1 and KAP1 differentially regulate the choice of inactive versus active X chromosomes.

The onset of random X chromosome inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. This process is known as X chromosome choice. Here, we show that RIF1 and KAP1 are two fundamental factors for the definition of this transcriptional asymmetry. We found that at the onset of differentiation of mouse embryonic stem cells (mESCs), biallelic up-regulation of the long non-coding RNA Tsix weakens the symmetric association of RIF1 with the Xist promoter. The Xist allele maintaining the association with RIF1 goes on to up-regulate Xist RNA expression in a RIF1-dependent manner. Conversely, the promoter that loses RIF1 gains binding of KAP1, and KAP1 is required for the increase in Tsix levels preceding the choice. We propose that the mutual exclusion of Tsix and RIF1, and of RIF1 and KAP1, at the Xist promoters establish a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X-chromosome state.

Oct1 regulates trophoblast development during early mouse embryogenesis.

Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

Subsets of cloned mouse embryos and their non-random relationship to development and nuclear reprogramming.

An important question in oocyte-mediated nuclear reprogramming is whether gene expression of the donor nucleus changes randomly or follows a pattern. Since cloned embryos are very heterogeneous and arrest frequently during preimplantation development, a random scenario is generally accepted. In the present study, we resolve the heterogeneity of cumulus cell-derived mouse clones by recognizing structured subsets, and we analyze their relationship to reprogramming of donor nuclei. We utilize live cell imaging of the Oct4 promoter-driven GFP transgene to resolve the populations of cloned and ICSI-fertilized morulae, and we sort them both into three subsets based on different GFP expression. Functionally, subsets of cloned but not ICSI morulae form blastocysts and ES cells proportional to Oct4-GFP expression. Regulatively, the subsets of cloned morulae are characterized by small differences of transcript level for the pluripotency-associated genes Oct4, Nanog and Sox2. Small differences of the level of select mRNAs across subsets suggest a uniform rather than random course of reprogramming from the morula stage on. Since these small differences correspond with substantial differences in developmental competence, we propose that developmental potential of clones relates to levels of gene expression in a different way than fertilized embryos.

Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus.

A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction.

Chromosome stability differs in cloned mouse embryos and derivative ES cells.

The mechanisms that have evolved to maintain genome stability during cell cycle progression are challenged when a somatic cell nucleus is placed in a meiotic environment such as the ooplasm. Chromosomal spindle aberrations ensue in the majority of reconstructed oocytes within 2 h of transplantation, but it is not known if they recover or persist with the onset of embryonic divisions. We analyzed the chromosomal spindles and the karyotype of cumulus cell-derived mouse clones through the initial and hence most critical mitoses. Cloned embryos start out with less aneuploidy than fertilized embryos but surpass them after ES cell derivation, as measured by frequencies of chromosome trisomies and structural rearrangements. Despite the limited proportion of cloned mouse embryos that reach late gestation, a phenotypic mutation lacking a karyotypic mark was found in a newborn mouse cloned in 2002 and has been inherited since by its offspring. These data concur with a prevalent epigenetic, rather than genetic, basis for cloned embryo failure, but they also warn against the temptation to think that all conditions of clones are epigenetic and recover during gametogenesis. The cloning procedure is defenseless (no matter how technically refined) towards pre-existing or induced subchromosomal mutations that are below the experimental detection limit of the cytogenetic assay.

The caudal-related protein cdx2 promotes trophoblast differentiation of mouse embryonic stem cells.

Besides holding great promise in clinics, embryonic stem (ES) cells represent a valuable tool for studying regulation of early developmental processes, such as cell differentiation in preimplantation embryos. The caudal-related homeobox protein Cdx2 is a transcriptional regulator essential for trophoblast lineage, functioning as early as implantation. Using an inducible system, we show that gain of Cdx2 function in ES cells triggers trophoblast-like morphological differentiation, accompanied by ploidy increase, onset of expression of trophoblast-specific markers, and loss of pluripotency-associated gene expression. These data provide an insight into the genetic network that controls lineage specification and functioning in early mammalian development.

Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments.

A prevailing view of cloning by somatic-cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.

Nanog: a new recruit to the embryonic stem cell orchestra.

In this issue of Cell, and add a new transcriptional operating system to the known Oct4 and Stat3 systems required for early embryonal stem cell potency and self-renewal. Nanog, a homeobox transcription factor, plays a crucial role in the second embryonic cell fate specification following formation of the blastocyst.