Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection.
Ontology highlight
ABSTRACT:
SUBMITTER: Sousa-Nunes R
PROVIDER: S-EPMC403803 | biostudies-literature | 2003 Dec
REPOSITORIES: biostudies-literature
Publications
Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection.
Sousa-Nunes Rita R Rana Amer Ahmed AA Kettleborough Ross R Brickman Joshua M JM Clements Melanie M Forrest Alistair A Grimmond Sean S Avner Philip P Smith James C JC Dunwoodie Sally L SL Beddington Rosa S P RS
Genome research 20031112 12
This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node-tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence wa ...[more]
Similar Datasets
Project description:Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.
| S-EPMC3237556 | biostudies-literature
Project description:BackgroundThe polo-like kinases (Plks) are a group of serine/threonine kinases which have roles in many aspects of cellular function including the regulation of mitotic activity and cellular stress responses. This study focuses on Plk4, the most divergent member of the Plk family, which is necessary for proper cellular proliferation. More specifically, alterations in Plk4 levels cause significantly adverse mitotic defects including abnormal centrosome duplication and aberrant mitotic spindle formation. We sought to clarify the effect of reduced Plk4 levels on the cell by examining transcript profiles of Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs). Subsequently, the levels of several key proteins involved in the DNA damage response were examined.Results143 genes were found to be significantly up-regulated in the heterozygous MEFs compared to their wild-type counterparts, while conversely, 9 genes were down-regulated. Numerous genes with increased transcript levels in heterozygous MEFs were identified to be involved in p53-dependent pathways. Furthermore, examination of the promoter regions of all up- and down-regulated genes revealed that the majority contained putative p53 responsive elements. An analysis of transcript levels in MEFs after exposure to either ionizing or ultraviolet radiation revealed a significant change between wild type and heterozygous MEFS for Plk4 transcript levels upon only UV exposure. Furthermore, changes in protein levels of several important cell check-point and apoptosis regulators were examined, including p53, Chk1, Chk2, Cdc25C and p21. In heterozygous MEFs, p53, p21 and Chk2 protein levels were at significantly higher levels. Furthermore, p53 activity was increased 5 fold in the Plk4 heterozygous MEFs.ConclusionGlobal transcript profiles and levels of key proteins involved in cellular proliferation and DNA damage pathways were examined in wild-type and Plk4 heterozygous MEFs. It was determined that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. We propose a model in which reduced Plk4 levels invoke an increase in p53 levels that leads to the aforementioned changes in global transcription profiles.
| S-EPMC2727538 | biostudies-literature
Project description:Wnt signalling is one of the fundamental cell communication systems operating in the embryo and the collection of 19 Wnt and 10 Frizzled (Fzd) receptor genes (in mouse and human) represent just part of a complex system to be unravelled. Here we present a spatially comprehensive set of data on the 3D distribution of Wnt and Fzd gene expression patterns at a carefully selected single stage of mouse development. Overviews and selected features of the patterns are presented and the full 3D data set, generated by fully described probes, is available to the research community through the Edinburgh Mouse Atlas of Gene Expression. In addition to being comprehensive, the data set has been generated and recorded in a consistent manner to facilitate comparisons between gene expression patterns with the capacity to generate matching virtual sections from the 3D representations for specific studies. Expression patterns in the left forelimb were selected for more detailed comparative description. In addition to confirming the previously published expression of these genes, our whole embryo and limb bud analyses significantly extend the data in terms of details of the patterns and the addition of previously undetected sites of expression. Our focussed analysis of expression domains in the limb, defined by just two gene families, reveals a surprisingly high degree of spatial complexity and underlines the enormous potential for local cellular interactions that exist within an emerging structure. This work also highlights the use of OPT to generate detailed high-quality, spatially complex expression data that is readily comparable between specimens and can be reviewed and reanalysed as required for specific studies. It represents a core set of data that will be extended with additional stages of development and through addition of potentially interacting genes and ultimately other cross-regulatory communication pathways operating in the embryo.
| S-EPMC2452985 | biostudies-literature
Project description:Nephronophthisis-related ciliopathies (NPHP-RC) are a group of inherited genetic disorders that share a defect in the formation, maintenance or functioning of the primary cilium complex, causing progressive cystic kidney disease and other clinical manifestations. Mutations in centrosomal protein 164 kDa (CEP164), also known as NPHP15, have been identified as a cause of NPHP-RC. Here we have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) to perform immunohistochemistry studies on human embryonic and foetal tissues to determine the expression patterns of CEP164 during development. Notably expression is widespread, yet defined, in multiple organs including the kidney, retina and cerebellum. Murine studies demonstrated an almost identical Cep164 expression pattern. Taken together, these data support a conserved role for CEP164 throughout the development of numerous organs, which, we suggest, accounts for the multi-system disease phenotype of CEP164-mediated NPHP-RC.
| S-EPMC6986751 | biostudies-literature
Project description:BackgroundIt has been a long-standing biological challenge to understand the molecular regulatory mechanisms behind mammalian ageing. Harnessing the availability of many ageing microarray datasets, a number of studies have shown that it is possible to identify genes that have age-dependent differential expression (DE) or differential variability (DV) patterns. The majority of the studies identify "interesting" genes using a linear regression approach, which is known to perform poorly in the presence of outliers or if the underlying age-dependent pattern is non-linear. Clearly a more robust and flexible approach is needed to identify genes with various age-dependent gene expression patterns.ResultsHere we present a novel model selection approach to discover genes with linear or non-linear age-dependent gene expression patterns from microarray data. To identify DE genes, our method fits three quantile regression models (constant, linear and piecewise linear models) to the expression profile of each gene, and selects the least complex model that best fits the available data. Similarly, DV genes are identified by fitting and comparing two quantile regression models (non-DV and the DV models) to the expression profile of each gene. We show that our approach is much more robust than the standard linear regression approach in discovering age-dependent patterns. We also applied our approach to analyze two human brain ageing datasets and found many biologically interesting gene expression patterns, including some very interesting DV patterns, that have been overlooked in the original studies. Furthermore, we propose that our model selection approach can be extended to discover DE and DV genes from microarray datasets with discrete class labels, by considering different quantile regression models.ConclusionIn this paper, we present a novel application of quantile regression models to identify genes that have interesting linear or non-linear age-dependent expression patterns. One important contribution of this paper is to introduce a model selection approach to DE and DV gene identification, which is most commonly tackled by null hypothesis testing approaches. We show that our approach is robust in analyzing real and simulated datasets. We believe that our approach is applicable in many ageing or time-series data analysis tasks.
| S-EPMC2788368 | biostudies-literature
Project description:Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.
| S-EPMC3273828 | biostudies-literature
Project description:Ewing's sarcoma is a rare bone tumor that affects children and adolescents. We have recently succeeded to induce Ewing's sarcoma-like small round cell tumor in mice by expression of EWS-ETS fusion genes in murine embryonic osteochondrogenic progenitors. The Ewing's sarcoma precursors are enriched in embryonic superficial zone (eSZ) cells of long bone. To get insights into the mechanisms of Ewing's sarcoma development, gene expression profiles between EWS-FLI1-sensitive eSZ cells and EWS-FLI1-resistant embryonic growth plate (eGP) cells were compared using DNA microarrays. Gene expression of eSZ and eGP cells (total, 30 samples) was evaluated with or without EWS-FLI1 expression 0, 8 or 48 h after gene transduction. Our data provide useful information for gene expression responses to fusion oncogenes in human sarcoma.
| S-EPMC4535656 | biostudies-literature
Project description:The association between breast cancer risk defined by the Tyrer-Cuzick score (TC) and disease prognosis is not well established. Here, we investigated the relationship between 5-year TC and disease aggressiveness and then characterized underlying molecular processes. In a case-only study (n = 2474), we studied the association of TC with molecular subtypes and tumor characteristics. In a subset of patients (n = 672), we correlated gene expression to TC and computed a low-risk TC gene expression (TC-Gx) profile, that is, a profile expected to be negatively associated with risk, which we used to test for association with disease aggressiveness. We performed enrichment analysis to pinpoint molecular processes likely to be altered in low-risk tumors. A higher TC was found to be inversely associated with more aggressive surrogate molecular subtypes and tumor characteristics (P < .05) including Ki-67 proliferation status (P < 5 × 10-07 ). Our low-risk TC-Gx, based on the weighted sum of 37 expression values of genes strongly correlated with TC, was associated with basal-like (P < 5 × 10-13 ), HER2-enriched subtype (P < 5 × 10-07 ) and worse 10-year breast cancer-specific survival (log-rank P < 5 × 10-04 ). Associations between low-risk TC-Gx and more aggressive molecular subtypes were replicated in an independent cohort from The Cancer Genome Atlas database (n = 975). Gene expression that correlated with low TC was enriched in proliferation and oncogenic signaling pathways (FDR < 0.05). Moreover, higher proliferation was a key factor explaining the association with worse survival. Women who developed breast cancer despite having a low risk were diagnosed with more aggressive tumors and had a worse prognosis, most likely driven by increased proliferation. Our findings imply the need to establish risk factors associated with more aggressive breast cancer subtypes.
| S-EPMC7818270 | biostudies-literature
Project description:BackgroundNatural selection can act on multiple genes in the same pathway, leading to polygenic adaptation. For example, adaptive changes were found to down-regulate six genes involved in ergosterol biosynthesis-an essential pathway targeted by many antifungal drugs-in some strains of the yeast Saccharomyces cerevisiae. However, the impact of this polygenic adaptation on metabolite levels was unknown. Here, we performed targeted mass spectrometry to measure the levels of eight metabolites in this pathway in 74 yeast strains from a genetic cross.ResultsThrough quantitative trait locus (QTL) mapping we identified 19 loci affecting ergosterol pathway metabolite levels, many of which overlap loci that also impact gene expression within the pathway. We then used the recently developed v-test, which identified selection acting upon three metabolite levels within the pathway, none of which were predictable from the gene expression adaptation.ConclusionsThese data showed that effects of selection on metabolite levels were complex and not predictable from gene expression data. This suggests that a deeper understanding of metabolism is necessary before we can understand the impacts of even relatively straightforward gene expression adaptations on metabolic pathways.
| S-EPMC8482673 | biostudies-literature
Project description:BackgroundUnderstanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs.ResultsAn initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.ConclusionES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.
| S-EPMC2656490 | biostudies-literature