We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

The character of a cell is defined by its constituent proteins, which are the result of specific patterns of gene expression. Crucial determinants of gene expression patterns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by recognizing the sequence of DNA bases in their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the structure of chromatin, that leads to the assembly of an active transcription complex (e.g., Cosma et al. 1999). But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity, as the transcriptional potential of a genome can become restricted in a stable manner during development. The constraints imposed by developmental history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi 2001). A “transcription factors only” model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors. Although many aspects of expression can be reprogrammed in this way (Gurdon 1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory. The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zygote from which it is descended (mammalian B and T cells being an obvious exception). This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation. Processes less irrevocable than mutation fall under the umbrella term “epigenetic” mechanisms. A current definition of epigenetics is: “The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al. 1996). There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx) protein complexes. (Histone modification has some attributes of an epigenetic process, but the issue of heritability has yet to be resolved.) This review concerns DNA methylation, focusing on the generation, inheritance, and biological significance of genomic methylation patterns in the development of mammals. Data will be discussed favoring the notion that DNA methylation may only affect genes that are already silenced by other mechanisms in the embryo. Embryonic transcription, on the other hand, may cause the exclusion of the DNA methylation machinery. The heritability of methylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development. Indeed, the possibility will be discussed that DNA methylation and Pc-G/trx may represent alternative systems of epigenetic memory that have been interchanged over evolutionary time. Animal DNA methylation has been the subject of several recent reviews (Bird and Wolffe 1999; Bestor 2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al. (2000), Martienssen and Colot (2001), and Matzke et al. (2001).

/pdf/dna-methylation-patterns-and-epigenetic-memory-2xnqjp4ije.pdf

DNA methylation patterns and epigenetic memory

A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application

http://www.westlaboratory.org/wp-content/uploads/2011/05/Okita-2007-Generation-of-germline-competent-induced-pluripotent-stem-cells.pdf

Generation of germline-competent induced pluripotent stem cells

Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

/pdf/characterization-of-micrornas-in-serum-a-novel-class-of-3urw6sycxl.pdf

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.

Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.

Blimp1 is a critical determinant of the germ cell lineage in mice

Germ cell fate in mice is induced in proximal epiblast cells by the extra-embryonic ectoderm, and is not acquired through the inheritance of any preformed germ plasm. To determine precisely how germ cells are specified, we performed a genetic screen between single nascent germ cells and their somatic neighbours that share common ancestry. Here we show that fragilis, an interferon-inducible transmembrane protein, marks the onset of germ cell competence, and we propose that through homotypic association, it demarcates germ cells from somatic neighbours. Using single-cell gene expression profiles, we also show that only those cells with the highest expression of fragilis subsequently express stella, a gene that we detected exclusively in lineage-restricted germ cells. The stella positive nascent germ cells exhibit repression of homeobox genes, which may explain their escape from a somatic cell fate and the retention of pluripotency.

A molecular programme for the specification of germ cell fate in mice

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

The polycomb-group gene Ezh2 is required for early mouse development.

There has been much speculation on whether mammalian eggs with two male pronuclei can develop normally. Eggs with two female pronuclei can sometimes develop as far as the 25-somite stage1–3 but with only very meagre extraembryonic tissues2,3. We suggested that the genome undergoes specific imprinting during gametogenesis3 and that some paternal genes may be necessary for normal development of the extraembryonic tissues3,4, in which only the maternal X chromosome remains active5–9. However, the need for the maternal genome for development to term is not yet unequivocally established. The detailed study described here demonstrates that while between 40 and 50% of heterozygous reconstituted eggs with a male and a female pronucleus develop to term, none of the eggs with two male pronuclei does so. Furthermore, embryos in the latter case are very retarded, even though the trophoblast develops relatively well compared with embryos having two female pronuclei1–3. Our combined results indicate that while the paternal genome is essential for the normal development of extraembryonic tissues, the maternal genome may be essential for some stages of embryogenesis.

Sheila C. Barton

Papers

Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

Blimp1 is a critical determinant of the germ cell lineage in mice

A molecular programme for the specification of germ cell fate in mice

The polycomb-group gene Ezh2 is required for early mouse development.

Role of paternal and maternal genomes in mouse development