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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
Published on: February 22, 2016
LYAR Regulates Ribosomal Genes to Impact the First Lineage Differentiation During Mouse Preimplantation Development
Wenyao Zhou1, Yuchen Sun1, Xinglin Hu1
1Department of Histology and Embryology, Harbin Medical University, Harbin, China.
Abstract:
The transcription of ribosomal genes (rDNA) and precursor rRNA (pre-rRNA) processing are crucial biological processes in ribosome biogenesis. Yet, the specific impact of ribosome biogenesis on the first lineage differentiation during early mouse embryonic development remains unclear. Nucleolar proteinLy-1 antibody reactive clone (LYAR) can accelerate ribosome production, regulating rDNA transcription and pre-rRNA maturation. Our study employed immunofluorescence and quantitative polymerase chain reaction (qPCR) to investigate the presence and localization of LYAR during early mouse embryonic development. The interference fragments and mRNA of LYAR were separately injected into embryos at the pronuclear stage, and the embryo development was statistically analysed. The impact of regulating LYAR expression on the first lineage differentiation was determined by immunofluorescence staining, At the 2-cell stage, regulatory fragments and RFP mRNA were injected into a single blastomere. By immunofluorescence staining to determine the proportion of blastomeres with regulated LYAR expression contributing to the ICM. The influence of interfering with LYAR expression at the pronuclear stage on the overall level of newly synthesised rRNA was detected by EU staining. Finally, qPCR was used to further determine the impact of regulating LYAR expression on rDNA transcription and pre-rRNA processing. The results demonstrate that LYAR is detectable at various stages of preimplantation development. Importantly, as embryonic development progresses, LYAR gradually translocates from the cytoplasm and nucleus into the nucleolus. Knockdown of LYAR during the pronuclear stage results in a reduction in the number of ICM following the first lineage differentiation of embryonic development. Furthermore, modulating the expression of LYAR in individual blastomeres at the 2-cell stage affects their contribution to the ICM, altering the proportion of cells that contribute to the blastocyst. Our study hints a link between ribosome biogenesis and the first lineage differentiation, contributing to a better understanding of the mechanisms underlying mouse embryonic development.
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