Bmi1 is expressed in vivo in intestinal stem cells

Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of beta-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1(+) cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis.     

Epigenetic status of human embryonic stem cells

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.     

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.     

Current issues in mouse genome engineering

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.     

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.     

Dicer is essential for mouse development

To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.     

Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos

In blastocyst chimeras, embryonic stem (ES) cells contribute to embryonic tissues but not extraembryonic trophectoderm. Conditional activation of HRas1(Q61L) in ES cells in vitro induces the trophectoderm marker Cdx2 and enables derivation of trophoblast stem (TS) cell lines that, when injected into blastocysts, chimerize placental tissues. Erk2, the downstream effector of Ras-mitogen-activated protein kinase (MAPK) signaling, is asymmetrically expressed in the apical membranes of the 8-cell-stage embryo just before morula compaction. Inhibition of MAPK signaling in cultured mouse embryos compromises Cdx2 expression, delays blastocyst development and reduces trophectoderm outgrowth from embryo explants. These data show that ectopic Ras activation can divert ES cells toward extraembryonic trophoblastic fates and implicate Ras-MAPK signaling in promoting trophectoderm formation from mouse embryos.     

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.     

Functional analysis of secreted and transmembrane proteins critical to mouse development.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.     

Mutagenic insertion and chromosome engineering resource (MICER)

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.     

Cancer predisposition caused by elevated mitotic recombination in Bloom mice

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.     

Lgr5 marks cycling, yet long-lived, hair follicle stem cells

In mouse hair follicles, a group of quiescent cells in the bulge is believed to have stem cell activity. Lgr5, a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Here we show that Lgr5(+) cells comprise an actively proliferating and multipotent stem cell population able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time. Lgr5(+) progeny repopulate other stem cell compartments in the hair follicle, supporting the existence of a stem or progenitor cell hierarchy. By marking Lgr5(+) cells during trafficking through the lower outer root sheath, we show that these cells retain stem cell properties and contribute to hair follicle growth during the next anagen. Expression analysis suggests involvement of autocrine Hedgehog signaling in maintaining the Lgr5(+) stem cell population.     

DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal

The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.