The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to ...the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named
Zscan4a-Zscan4f, three –
Zscan4c,
Zscan4d, and
Zscan4f – encode full-length ORFs with 506 amino acids.
Zscan4d is a predominant transcript at the late 2-cell stage, whereas
Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any
Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development.
To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable ...tetracycline-repressible locus. Of the 50 TFs,
Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by
Esx1,
Sox9,
Tcf3,
Klf4, and
Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice.
Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression ...profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.
We previously reported that
Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput
...in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types.
Pou5f1 (
Oct4,
Oct3/
4) and
Krt8 (
EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed
Zscan4-like spotted expression pattern (spot-in-colony pattern;
Whsc2 and
Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern:
Zfp42/
Rex1,
Rest,
Atf4,
Pa2g4,
E2f2,
Nanog,
Dppa3/Pgc7/
Stella,
Esrrb, and
Fscn1). Among these genes,
Zfp42/
Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.
A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from ...embryonic and newborn organ libraries consisting of ~120,000 cDNA clones, which were initially re-arrayed into a set of ~11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers.
The choice of phthalates as plasticizers have been on the increase especially in household products. They are ubiquitous environmental pollutants due to their physical attribute. This study was ...carried out to determine the occurrence and level of phthalates in the groundwater in some regions of Delta State. Groundwater samples were collected from fourteen sampling points and analyzed using standard procedures. The obtained results showed that the concentration (µg/l) of six phthalate ester compounds present in the water samples was of the order: < 0.05 - 0.05 BBP, < 0.05 - 3.71 BEHP, < 0.05 - 0.54 DBP, < 0.05 - 0.55 DEP, < 0.05 - 0.13 DMP, and < 0.05 - 0.48 DnOP. BEHP was observed to be the major compound of the phthalate acid esters present in most sampling stations, whilst others, especially BBP, were found to be in low concentration and does not pose any immediate threat to human health. The presence of BEHP in most samples from different locations suggests an inflow of the phthalate to underground water, hence it becomes imperative for continuous monitoring and a call to various governments and environmental regulatory agencies to establish standards for phthalate esters in order to monitor its presence in the environment.
A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250000 ESTs, representing approximately 39000 unique transcripts. The cDNA libraries, ...enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells.
Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7-10% of all ...TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.
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