Cdkn1c (p57Kip2) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7.

Background: Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.

220-plex microRNA expression profile of a single cell.

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers.

Maternal microRNAs are essential for mouse zygotic development.

MicroRNAs (miRNAs) have important roles in diverse cellular processes, but little is known about their identity and functions during early mammalian development. Here, we show the effects of the loss of maternal inheritance of miRNAs following specific deletion of Dicer from growing oocytes. The mutant mature oocytes were almost entirely depleted of all miRNAs, and they failed to progress through the first cell division, probably because of disorganized spindle formation.

The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells.

The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2.

Generation of stella-GFP transgenic mice: a novel tool to study germ cell development.

The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages.

MicroRNA expression profiling of single whole embryonic stem cells.

MicroRNAs (miRNAs) are a class of 17-25 nt non-coding RNAs that have been shown to have critical functions in a wide variety of biological processes during development. Recently developed miRNA microarray techniques have helped to accelerate research on miRNAs. However, in some instances there is only a limited amount of material available for analysis, which requires more sensitive techniques that can preferably work on single cells.

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

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.

Analysis of imprinted murine Peg3 locus in transgenic mice.

Peg3 is an imprinted gene exclusively expressed from the paternal allele. It encodes a C(2)H(2) type zinc-finger protein and is involved in maternal behavior. It is important for TNF-NFkB signaling and p53-mediated apoptosis.

Stella is a maternal effect gene required for normal early development in mice.

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice.

Specification of germ cell fate in mice.

An early fundamental event during development is the segregation of germ cells from somatic cells. In many organisms, this is accomplished by the inheritance of preformed germ plasm, which apparently imposes transcriptional repression to prevent somatic cell fate. However, in mammals, pluripotent epiblast cells acquire germ cell fate in response to signalling molecules.

A molecular programme for the specification of germ cell fate 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.

Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers.

The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues.