Biomedical subjects

Tilo Kunath

Publications and source records attributed to Tilo Kunath.

7 recordsLinked to original sources

PubMed2006

p53-Independent apoptosis disrupts early organogenesis in embryos lacking both ataxia-telangiectasia mutated and Prkdc.

The ataxia-telangiectasia mutated (ATM) protein and the nonhomologous end-joining (NHEJ) pathway play crucial roles in sensing and repairing DNA double-strand breaks in postnatal cells. However, each pathway is dispensable for early embryogenesis. Loss of both ATM and Prkdc/Ku is synthetically lethal, but neither the developmental processes perturbed nor the mechanisms of lethality have been determined by previous reports. Here, we show that ATM and Prkdc collaborate to maintain genomic stability during gastrulation and early organogenesis, a period of rapid proliferation and hypersensitivity to DNA damage. At E7.5 to E8.5, ATM(-/-)Prkdcscid/scid embryos displayed normal proliferation indices but exhibited excessive apoptosis and elevated expression of Ser15-phosphorylated p53. Thus, this crucial regulatory residue of p53 can be phosphorylated in the absence of ATM or Prkdc. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that apoptosis is p53 independent in these in ATM(-/-)Prkdcscid/scid embryos. Because mice with combined disruptions of ATM and other NHEJ components (ligase IV, Artemis) are viable, our data suggest a novel NHEJ-independent function for Prkdc/Ku that is required to complete early embryogenesis in the absence of ATM.

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PubMed2006

Mouse trophoblast stem cells.

The trophectoderm is one of the earliest cell types to differentiate in the forming mammalian embryo. It is responsible for the initial implantation and the formation of the trophoblast components of the placenta, an organ essential for nutrient and waste exchange between the fetus and its mother. The trophoblast can be modeled in vitro using trophoblast stem cells. Trophoblast stem cells require fibroblast growth factor (FGF)4, heparin, and contact with embryonic fibroblasts, or fibroblast-conditioned medium. They grow as tight epithelial colonies, which express markers of the early trophectoderm and have been shown to contribute to all of the components of the placenta through chimera studies. These cells can be passaged indefinitely and can be differentiated by removal of FGF4 and fibroblasts and will express genetic markers of later placental cell types. This chapter will discuss the initial derivation of trophoblast stem cells from the blastocyst stage, maintenance, differentiation, flow cytometry and transfection techniques that can be used with these cells.

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PubMed2005

Phenotypic analyses of mouse embryos with ubiquitous expression of Oct4: effects on mid-hindbrain patterning and gene expression.

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid-hindbrain patterning and produced a strong up-regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down-regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse.

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PubMed2005

Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.

The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages, respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst -extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.

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PubMed2003

Transcriptome analysis of mouse stem cells and early embryos.

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

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PubMed2003

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype.

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.

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PubMed2002

Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity.

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.

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