Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse embryo to assess gene expression during preimplantation mouse development

ABSTRACT: Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.

ORGANISM(S): Mus musculus

SUBMITTER: Richard Schultz

PROVIDER: E-GEOD-1749 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcript profiling during preimplantation mouse development.

Zeng Fanyi F Baldwin Don A DA Schultz Richard M RM

Developmental biology 20040801 2

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular proces ...[more]

PMID: 15282163

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Project description:Dramatic changes in chromatin structure and histone modification occur during oocyte growth, as well as a global cessation of transcription. The role of histone modifications in these processes is poorly understood. We report the effect of conditionally deleting Hdac1 and Hdac2 on oocyte development. Deleting either gene has little or no effect on oocyte development, whereas deleting both genes results in follicle development arrest at the secondary follicle stage. This developmental arrest is accompanied by substantial perturbation of the transcriptome and a global reduction in transcription even though histone acetylation is markedly increased. There is no apparent change in histone repressive marks but there is a pronounced decrease in histone H3K4 methylation, an activating mark. The decrease in H3K4 methylation is likely due to increased expression of Kdm5b because RNAi-mediated targeting of Kdm5b in double mutant oocytes results in an increase in H3K4 methylation. An increase in TRP53 acetylation also occurs in mutant oocytes and may contribute to the observed increased incidence of apoptosis. Taken together, these results suggest seminal roles of acetylation of histone and non-histone proteins in oocyte development. We used microarrays to detail the global programme of gene expression underlying oocyte development and identified distinct classes of regulated genes during this process. Total RNA was extracted from 80 oocytes isolated from mice 12 days-of-age using the PicoPure RNA kit (Arcturus), amplified with the Ovation Pico WTA system (NuGen), and then fragmented and labeled with the FL-OvationTM cDNA Biotin Module V2 (NuGen). Four independent biological replicates were hybridized to Affymetrix GeneChip Mouse 1.1 ST microarrays (http://www.affymetrix.com/). Raw microarray data were analyzed as previously described using MAS5, GeneSpring v7, SAM and EASE software (56). A 1.4-fold cutoff was used for EASE analysis; four biological replicates provide sufficient statistical power and confidence to detect a 1.4-fold change in transcript abundance

Project description:The transformation of a fertilized oocyte into a multicellular embryo comprised of differentiated cells depends on the processing of genetic orders, first in the form of transcripts and then of proteins. Our current understanding of mouse development has greatly benefited from the analysis of transcripts as proxy for the proteins. However, the hexagonal-shaped free ribosomes that translate mRNAs into proteins are scarce during cleavage and do not become abundant until the morula-blastocyst stage. How accurate, then, is an understanding of mouse and more in general mammalian development that uses the analysis of transcripts as proxy for the proteins? This led us in this study to measure the proteome of seven preimplantation mouse stages (B6C3F1 x CD1), from oocyte to blastocyst, using 'spike-in' SILAC technology combined with high accuracy mass spectrometry (LTQ Orbitrap and Q-Exactive). The relative abundances of embryonic proteins were compared and contrasted with those of transcripts measured by RNA sequencing. A total of 6976 proteins were detected in at least the spike (precondition for determining the heavy/light peptide ratios in oocytes or embryos). Among these 6976 proteins, 4991 proteins were present in all developmental stages, and 1893 proteins were present in all replicates. Our tandem approach uncovered: 1) the different abundance profiles of proteome and transcriptome during the time of preimplantation development; 2) the different reciprocal associations of developmental stages (dendrograms) on the protein level as compared to the mRNA level; and 3) the different gene ontology terms overrepresented among the proteins that increase or decrease across adjacent stages, compared to mRNAs. In essence, the elaborate phasing of embryonic gene expression that has been known for mRNAs leaves an unsuspected footprint on the protein products of those mRNAs. This information facilitates the analysis of mammalian development in two important ways. First, it provides new insight into the regulation of the transition from the differentiated oocyte into the embryo, by identifying the subsets of proteins that accompany the passage from one embryonic stage to the next, which are likely composed of important regulators of the morphogenetic transitions. Second, our analysis provides a reference to determine the degree of ‘embryoness’ of entities produced via assisted reproductive technologies, cloning by somatic cell nuclear transfer, and even artificial gametes.

Dataset Information

Transcription profiling of mouse embryo to assess gene expression during preimplantation mouse development

Publications

Transcript profiling during preimplantation mouse development.

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