Jane Taylor

Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells

We report here that the formation of heterochromatin in cell nuclei during mouse development is characterised by dynamic changes in the epigenetic modifications of histones. Our observations reveal that heterochromatin in mouse preimplantation embryos is in an immature state that lacks the constitutive heterochromatin markers histone H4 trimethyl Lys20 (H4K20me3) and chromobox homolog 5 (HP1α, also known as CBX5). Remarkably, these somatic heterochromatin hallmarks are not detectable – except in mural trophoblast – until mid-gestation, increasing in level during foetal development. Our results support a developmentally regulated connection between HP1α and H4K20me3. Whereas inner cell mass (ICM) and epiblast stain negative for H4K20me3 and HP1α, embryonic stem (ES) cell lines, by contrast, stain positive for these markers, indicating substantial chromatin divergence. We conclude that H4K20me3 and HP1α are late developmental epigenetic markers, and slow maturation of heterochromatin in tissues that develop from ICM is ectopically induced during ES cell derivation. Our findings suggest that H4K20me3 and HP1α are markers for cell type commitment that can be triggered by developmental or cell context, independently of the differentiation process.

Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage.

The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.

Rat eggs cannot wait: Spontaneous exit from meiotic metaphase‐II arrest

Mammalian eggs await fertilisation while arrested at the second metaphase stage of meiotic division. A network of signalling pathways enables the establishment and maintenance of this metaphase‐II arrest. In the absence of fertilisation, mammalian eggs can spontaneously exit metaphase II when parthenogenetically stimulated, or sometimes without any obvious stimulation. Ovulated rat eggs abortively release from metaphase‐II arrest once removed from egg donors. Spontaneously activated rat eggs extrude the second polar body and proceed to the so‐called metaphase III‐‘like’ stage, with clumps of condensed chromatin scattered in the egg cytoplasm. It is still unclear what makes rat eggs susceptible to spontaneous activation; however, a vague picture of the signalling pathways involved in the process of spontaneous activation is beginning to emerge. Such cell cycle instability is one of the major reasons why it is more difficult to establish nuclear transfer in the rat. This review examines the known predisposing factors and biochemical mechanisms involved in spontaneous activation. The strategies used to prevent spontaneous metaphase‐II release in rat eggs will also be discussed. Mol. Reprod. Dev. 78:795–807, 2011.

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8–16 cell stage. Attempts to clone oocyte‐specific 5′ regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi‐mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. Mol. Reprod. Dev. 76: 501–513, 2009.

Somatic cell nuclear transfer: origins, the present position and future opportunities

Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce ‘induced pluripotent stem cells’ is providing revolutionary new opportunities in research and cell therapy.

A decade of progress since the birth of Dolly

The greatest effect of the birth Dolly, the first cloned animal derived from an adult, has been in prompting biologists to consider ways of reprogramming adult nuclei to a pluripotent state directly. The first procedure depends upon use of viral vectors to introduce selected transcription factors, but this procedure is slow and very inefficient. Research in our laboratory has demonstrated that exposure of differentiated nuclei to an extract of embryo stem cells induces expression of key pluripotency genes within 8 h, suggesting that it may be possible to identify and use other factors to enhance direct reprogramming. A study of mechanisms that bring about changes in DNA methylation in early sheep embryos identified a developmental isoform of Dnmt1, the expression of which was limited to early stages of pregnancy. Reduction in the level of transcript of this isoform at the time of fertilisation caused sheep embryo development to cease at the early morula stage, revealing a key role for the isoform that remains to be characterised. The ability to obtain pluripotent cells from specific patients is providing important new opportunities to study inherited diseases when the causative mutation is not known. The initial objective of this research is not cell therapy, but to use cells with the characteristics of those in a patient who has inherited the disease to establish a high-throughput screen to identify drugs that are able to prevent progression of the symptoms of the disease. Research is in progress with cells from patients with amyotropic lateral sclerosis.

Quantitative Evaluation of Reference Genes for Real‐Time PCR During In Vitro Maturation of Ovine Oocytes

Quantitative reverse transcription PCR (RT-qPCR) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB, GAPDH, H2AFZ, HPRT1, PPIA, SDHA, TUBB and YWHAZ and for sheep oocyte RT-qPCR before and after in vitro maturation. We have also compared different cDNA priming strategies using random hexamers or oligo-dT. GeNorm analysis of the results identified the most reliable genes for normalization to be SDHA, TUBB and PPIA when oocyte cDNA was made with random hexamers, and YWHAZ, TUBB and SDHA when oligo-dT primers were used (H2AFZ and HPRT1 were excluded from the geNorm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH, which are the conventional housekeeping genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed.

What are the limits to cell plasticity

It is now well established that the fate of a somatic cell is not fixed rigidly and that there is a significant degree of cell plasticity. The term plasticity refers to the opportunity to change differentiated cells from one cell type to another. Over the past 25 years a series of papers have each demonstrated that plasticity is wider than had previously been under-stood [1-4]. An exciting recent article by Thomas Vierbuchen and colleagues at Stanford University extended that series by describing a method for directly re-programming mouse fibroblast cells into neurons without the need to generate a stem cell intermediary [5].Vierbuchen and colleagues isolated fibroblasts from embryos (mouse em-bryonic fibroblasts – MEFs) and postna-tal tail-tips from transgenic (TauEGFP knock-in) mice. TauEGFP reporter mice express the green fluorescent protein in post-mitotic neurons thus allowing visualisation of motor and sensory neuronal cells as they develop, aiding the identification of successful repro-gramming. A drug-inducible lentiviral system containing 19 candidate genes selected as being either expressed in neural cell types or essential for repro-gramming to pluripotency was used to reprogramme fibroblast to a neuronal state. From this group five factors that were able to evoke a neuronal pheno-type were

Stem cells: Primates join the club

Researchers have achieved the testing goal of generating embryonic stem cells from the cells of an adult primate. The procedure used could provide insights into a variety of diseases, if it can be applied in humans.

Optimisation of a serum albumin removal protocol for use in a proteomic study to identify the protein biomarkers for silent gastric ulceration in horses

ABSTRACT Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted serum to identify the protein biomarkers for silent gastric ulceration. Gastroscopy was used to identify gastric ulceration, and serum was obtained from horses with either a healthy gastric mucosa or gastric ulceration. Serum albumin was removed using the trichloroacetic acid (TCA) protein precipitation method, and this protocol was optimised by varying the concentration of TCA, type of organic solvents, ratio of serum to protein precipitation solution, and incubation times. Electrophoresis and image analysis were used to compare the amounts of albumin, immunoglobulins G (IgG), and protein degradation before and after TCA precipitation. The best protocol was chosen to remove albumin for a proteomic study (electrophoresis and mass spectrometry). The results revealed that protocol 2 (ratio of serum to solution 1:5, 10% TCA in acetone, and 90 min incubation) was the most efficient protocol to remove albumin (98%) and IgG heavy (80%) and light (98%) chains without degrading other proteins. After electrophoresis and mass spectrometry analysis, KRT1, KRT6A and KRT18 were identified as potential markers for silent gastric ulceration.