MED20 is essential for early embryogenesis and regulates NANOG expression

in Reproduction
Correspondence should be addressed to W Cui or J Mager; Email: or
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Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.


    Society for Reproduction and Fertility

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    (A) Schematic of Med20 gene, CRISPR-Cas9-mediated deletion, genotyping primers for WT allele and Mut allele, RT-PCR primers (flanking intron2, which is 5691 bp) and three different siRNAs. F, forward; R, reverse. (B) Representative genotyped embryos at E7.5. (C) Representative genotyped embryos at E3.5. (D) The outgrowths produced by different genotypes. Outgrowths from WT and Het displayed a distinct ICM colony (red dashed line) surrounded by robustly proliferating trophoblast cells (blue dashed line). Scale bars, 100 μm.

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    Knockout of Med20 did not affect apoptosis index or ICM/TE lineage specification. Blastocysts in this experiment were flushed at E3.5, and then cultured overnight before fixation and immunofluorescence. Blastocysts of all genotypes showed low apoptosis index (TRP53 as the marker) and that ICM cells were tightly arranged with robust expression of OCT4, while TE cells were uniformly arranged with specific expression of CDX2. Scale bar, 50 μm.

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    Knockout of Med20 did not affect the expression or localization of either SOX17 (marker of primitive endoderm) or CDX2 (marker of trophectoderm); however, KO of Med20 led to severe ectopic expression of NANOG (marker of epiblast) in outside CDX2-positive TE cells. Blastocysts in this experiment were flushed at E3.5, and then cultured overnight before fixation and immunofluorescence. Scale bar, 50 μm.

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    (A) Expression pattern of Med20 in WT preimplantation embryos. Actb was used as loading control. Oo, metaphase II oocyte; Zy, zygote; 2C, 2-cell embryo; 4/8C, mix of 4- and 8-cell stage embryos; Mo, morula; Blas, blastocyst. (B) Simultaneous extraction of both RNA and DNA from single blastocyst to perform both genotyping PCR and Med20 RT-PCR, confirming KO was successful. Actb was used as loading control. (C) Endogenous Med20 mRNA was significantly depleted by three distinct siRNAs after microinjection. (D) KD of Med20 using distinct siRNAs did not affect blastocyst formation or morphology, but significantly altered embryo outgrowth potential. Red and blue dashed lines indicate ICM colony and trophoblast cells, respectively. Control: scrambled siRNA. n, number of embryos; *, P < 0.05. Scale bars, 100 μm.

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    Knockdown of Med20 by three distinct siRNAs did not affect the expression or localization of either SOX17 (primitive endoderm marker) or CDX2 (trophectoderm marker), but resulted in ectopic expression of NANOG in CDX2-positive TE cells. Blastocysts in this experiment were harvested at 4 days post microinjection, and then fixed for immunofluorescence. Scale bar, 50 μm.


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