MED20 is essential for early embryogenesis and regulates NANOG expression

in Reproduction
Correspondence should be addressed to W Cui or J Mager; Email: wcui@umass.edu or jmager@vasci.umass.edu
Restricted access

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

Article Information

Metrics

All Time Past Year Past 30 Days
Abstract Views 94 94 94
Full Text Views 16 16 16
PDF Downloads 8 8 8

Altmetrics

Related Articles

Figures

  • View in gallery

    (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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    (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.

  • View in gallery

    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.

References

ApostolouEFerrariFWalshRMBar-NurOStadtfeldMCheloufiSStuartHTPoloJMOhsumiTKBorowskyML et al. 2013 Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming. Cell Stem Cell 12 699712. (https://doi.org/10.1016/j.stem.2013.04.013)

ArmantDR 2005 Blastocysts don’t go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells. Developmental Biology 280 260280. (https://doi.org/10.1016/j.ydbio.2005.02.009)

ArnoldSJRobertsonEJ 2009 Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nature Reviews Molecular Cell Biology 10 91103. (https://doi.org/10.1038/nrm2618)

BassalertCValverde-EstrellaLChazaudC 2018 Primitive endoderm differentiation: from specification to epithelialization. Current Topics in Developmental Biology 128 81104. (https://doi.org/10.1016/bs.ctdb.2017.12.001)

BeyerKSBeauchampRLLeeMFGusellaJFNaarAMRameshV 2007 Mediator subunit MED28 (Magicin) is a repressor of smooth muscle cell differentiation. Journal of Biological Chemistry 282 3215232157. (https://doi.org/10.1074/jbc.M706592200)

CaoZCareyTSGangulyAWilsonCAPaulSKnottJG 2015 Transcription factor AP-2gamma induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage. Development 142 16061615. (https://doi.org/10.1242/dev.120238)

CareyTSCaoZChoiIGangulyAWilsonCAPaulSKnottJG 2015 BRG1 governs nanog transcription in early mouse embryos and embryonic stem cells via antagonism of histone H3 lysine 9/14 acetylation. Molecular and Cellular Biology 35 41584169. (https://doi.org/10.1128/MCB.00546-15)

CarlstenJOZhuXGustafssonCM 2013 The multitalented Mediator complex. Trends in Biochemical Sciences 38 531537. (https://doi.org/10.1016/j.tibs.2013.08.007)

CarlstenJOZhuXLopezMDSamuelssonTGustafssonCM 2016 Loss of the Mediator subunit Med20 affects transcription of tRNA and other non-coding RNA genes in fission yeast. Biochimica et Biophysica Acta 1859 339347. (https://doi.org/10.1016/j.bbagrm.2015.11.007)

CockburnKRossantJ 2010 Making the blastocyst: lessons from the mouse. Journal of Clinical Investigation 120 9951003. (https://doi.org/10.1172/JCI41229)

CuiWDaiXMarchoCHanZZhangKTremblayKDMagerJ 2016a Towards functional annotation of the preimplantation transcriptome: an RNAi screen in mammalian embryos. Scientific Reports 6 37396. (https://doi.org/10.1038/srep37396)

CuiWPizzolloJHanZMarchoCZhangKMagerJ 2016b Nop2 is required for mammalian preimplantation development. Molecular Reproduction and Development 83 124131. (https://doi.org/10.1002/mrd.22600)

CuiWMagerJ 2018 Transcriptional regulation and genes involved in first lineage specification during preimplantation development. Advances in Anatomy Embryology and Cell Biology 229 3146. (https://doi.org/10.1007/978-3-319-63187-5_4)

DingNZhouHEstevePOChinHGKimSXuXJosephSMFriezMJSchwartzCEPradhanS et al. 2008 Mediator links epigenetic silencing of neuronal gene expression with x-linked mental retardation. Molecular Cell 31 347359. (https://doi.org/10.1016/j.molcel.2008.05.023)

FallathTKiddBNStillerJDavoineCBjorklundSMannersJMKazanKSchenkPM 2017 MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana. PLoS ONE 12 e0176022. (https://doi.org/10.1371/journal.pone.0176022)

FrumTRalstonA 2015 Cell signaling and transcription factors regulating cell fate during formation of the mouse blastocyst. Trends in Genetics 31 402410. (https://doi.org/10.1016/j.tig.2015.04.002)

HentgesKE 2011 Mediator complex proteins are required for diverse developmental processes. Seminars in Cell and Developmental Biology 22 769775. (https://doi.org/10.1016/j.semcdb.2011.07.025)

HuangYLiWYaoXLinQJYinJWLiangYHeinerMTianBHuiJWangG 2012 Mediator complex regulates alternative mRNA processing via the MED23 subunit. Molecular Cell 45 459469. (https://doi.org/10.1016/j.molcel.2011.12.022)

KageyMHNewmanJJBilodeauSZhanYOrlandoDAvan BerkumNLEbmeierCCGoossensJRahlPBLevineSS et al. 2010 Mediator and cohesin connect gene expression and chromatin architecture. Nature 467 430435. (https://doi.org/10.1038/nature09380)

KelleherRJ3rdFlanaganPMKornbergRD 1990 A novel mediator between activator proteins and the RNA polymerase II transcription apparatus. Cell 61 12091215. (https://doi.org/10.1016/0092-8674(90)90685-8)

KornbergRD 2005 Mediator and the mechanism of transcriptional activation. Trends in Biochemical Sciences 30 235239. (https://doi.org/10.1016/j.tibs.2005.03.011)

KurodaTTadaMKubotaHKimuraHHatanoSYSuemoriHNakatsujiNTadaT 2005 Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Molecular and Cellular Biology 25 24752485. (https://doi.org/10.1128/MCB.25.6.2475-2485.2005)

LathamKESolterDSchultzRM 1991 Activation of a two-cell stage-specific gene following transfer of heterologous nuclei into enucleated mouse embryos. Molecular Reproduction and Development 30 182186. (https://doi.org/10.1002/mrd.1080300303)

LeungCYZhuMZernicka-GoetzM 2016 Polarity in cell-fate acquisition in the early mouse embryo. Current Topics in Developmental Biology 120 203234. (https://doi.org/10.1016/bs.ctdb.2016.04.008)

LokkenAARalstonA 2016 The genetic regulation of cell fate during preimplantation mouse development. Current Topics in Developmental Biology 120 173202. (https://doi.org/10.1016/bs.ctdb.2016.04.006)

MarchoCCuiWMagerJ 2015 Epigenetic dynamics during preimplantation development. Reproduction 150 R109R120. (https://doi.org/10.1530/REP-15-0180)

MiaoYLGambiniAZhangYPadilla-BanksEJeffersonWNBernhardtMLHuangWLiLWilliamsCJ 2018 Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development in the mouse. Biology of Reproduction 98 449464. (https://doi.org/10.1093/biolre/ioy004)

MolotkovASorianoP 2018 Distinct mechanisms for PDGF and FGF signaling in primitive endoderm development. Developmental Biology 442 155161. (https://doi.org/10.1016/j.ydbio.2018.07.010)

MorganiSMMetzgerJJNicholsJSiggiaEDHadjantonakisAK 2018 Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning. Elife 7. (https://doi.org/10.7554/eLife.32839)

NicholsJZevnikBAnastassiadisKNiwaHKlewe-NebeniusDChambersIScholerHSmithA 1998 Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95 379391. (https://doi.org/10.1016/S0092-8674(00)81769-9)

NiwaH 2014 The pluripotency transcription factor network at work in reprogramming. Current Opinion in Genetics and Development 28 2531. (https://doi.org/10.1016/j.gde.2014.08.004)

NiwaHToyookaYShimosatoDStrumpfDTakahashiKYagiRRossantJ 2005 Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123 917929. (https://doi.org/10.1016/j.cell.2005.08.040)

ParkJMWernerJKimJMLisJTKimYJ 2001 Mediator, not holoenzyme, is directly recruited to the heat shock promoter by HSF upon heat shock. Molecular Cell 8 919. (https://doi.org/10.1016/S1097-2765(01)00296-9)

PaulSKnottJG 2014 Epigenetic control of cell fate in mouse blastocysts: the role of covalent histone modifications and chromatin remodeling. Molecular Reproduction and Development 81 171182. (https://doi.org/10.1002/mrd.22219)

Phillips-CreminsJESauriaMESanyalAGerasimovaTILajoieBRBellJSOngCTHookwayTAGuoCSunY et al. 2013 Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153 12811295. (https://doi.org/10.1016/j.cell.2013.04.053)

PiliszekAMadejaZEPlusaB 2017 Suppression of ERK signalling abolishes primitive endoderm formation but does not promote pluripotency in rabbit embryo. Development 144 37193730. (https://doi.org/10.1242/dev.156406)

QinJTakahashiYIsuzugawaKImaiMYamamotoSHiraiYImakawaK 2005 Regulation of embryo outgrowth by a morphogenic factor, epimorphin, in the mouse. Molecular Reproduction and Development 70 455463. (https://doi.org/10.1002/mrd.20225)

RalstonACoxBJNishiokaNSasakiHCheaERugg-GunnPGuoGRobsonPDraperJSRossantJ 2010 Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development 137 395403. (https://doi.org/10.1242/dev.038828)

RayonTMencheroSNietoAXenopoulosPCrespoMCockburnKCanonSSasakiHHadjantonakisAKde la PompaJL et al. 2014 Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst. Developmental Cell 30 410422. (https://doi.org/10.1016/j.devcel.2014.06.019)

RisleyMDClowesCYuMMitchellKHentgesKE 2010 The Mediator complex protein Med31 is required for embryonic growth and cell proliferation during mammalian development. Developmental Biology 342 146156. (https://doi.org/10.1016/j.ydbio.2010.03.019)

RochaPPScholzeMBleissWSchreweH 2010 Med12 is essential for early mouse development and for canonical Wnt and Wnt/PCP signaling. Development 137 27232731. (https://doi.org/10.1242/dev.053660)

RoddaDJChewJLLimLHLohYHWangBNgHHRobsonP 2005 Transcriptional regulation of nanog by OCT4 and SOX2. Journal of Biological Chemistry 280 2473124737. (https://doi.org/10.1074/jbc.M502573200)

StrumpfDMaoCAYamanakaYRalstonAChawengsaksophakKBeckFRossantJ 2005 Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132 20932102. (https://doi.org/10.1242/dev.01801)

TsaiKLTomomori-SatoCSatoSConawayRCConawayJWAsturiasFJ 2014 Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell 157 14301444. (https://doi.org/10.1016/j.cell.2014.05.015)

TutterAVKowalskiMPBaltusGAIourgenkoVLabowMLiEKadamS 2009 Role for Med12 in regulation of Nanog and Nanog target genes. Journal of Biological Chemistry 284 37093718. (https://doi.org/10.1074/jbc.M805677200)

WangKSenguptaSMagnaniLWilsonCAHenryRWKnottJG 2010 Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. PLoS ONE 5 e10622. (https://doi.org/10.1371/journal.pone.0010622)

WesterlingTKuuluvainenEMakelaTP 2007 Cdk8 is essential for preimplantation mouse development. Molecular and Cellular Biology 27 61776182. (https://doi.org/10.1128/MCB.01302-06)

WicklowEBlijSFrumTHirateYLangRASasakiHRalstonA 2014 HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLOS Genetics 10 e1004618. (https://doi.org/10.1371/journal.pgen.1004618)

YagiRKohnMJKaravanovaIKanekoKJVullhorstDDePamphilisMLBuonannoA 2007 Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 134 38273836. (https://doi.org/10.1242/dev.010223)

YinJWWangG 2014 The Mediator complex: a master coordinator of transcription and cell lineage development. Development 141 977987. (https://doi.org/10.1242/dev.098392)

PubMed

Google Scholar