61 Maternal-embryonic knockout of murine Med12 disrupts trophoblast differentiation

The Mediator complex plays a fundamental role in transcription regulation by facilitating interactions between transcription factors and RNA polymerase II. The core Mediator subunits are essential for cell survival. However, tetraploid complementation assays have shown that ablation of the Med12 subunit, which can transiently interact with the core complex, does not result in embryonic lethality until E7.5 (Rocha et al. 2010 Development 137, 2723–2731). As tetraploid complementation does not address the role of Med12 in pre-implantation embryos or extraembryonic tissues, we used Zp3-Cre transgenic mice to ablate Med12 from primary follicles. Surprisingly, oogenesis, folliculogenesis, and ovulation were unaffected; however, oocyte-specific Med12 ablation resulted in female sterility. To test the viability of Med12-depleted oocytes, 6- to 8-week-old Med12fl/fl Zp3-Cre females were mated with stud males (Med12+/y). Although both female (Med12Δ/+) and male (Med12Δ/y) embryos successfully implanted by E5.5, they were resorbed by E12.5. Histological analysis of Med12 null E9.5 implants showed increased populations of trophoblast giant cells (TGCs) and diminished spongiotrophoblast and labyrinth layers. Moreover, E7.5 implants demonstrated reduced CDX2 signal, suggesting dysregulation of trophoblast identity. To better understand how loss of maternal and embryonic Med12 impacts transcription during peri-implantation stages, individual male E7.5 embryos were collected from mutant (Med12fl/fl Zp3-Cre) and control (Med12fl/fl) dams for bulk RNA-seq. Embryos were dissected into the extraembryonic ectoderm (EEE; n = 3 controls, 2 mutants), ectoplacental cone (EPC; n = 4 controls, 3 mutants), and epiblast (EMB; n = 4 controls, 4 mutants). Female embryos were excluded to avoid confounding effects of X chromosome inactivation. Loss of Med12 led to dysregulation of 178, 447, and 314 genes in the EPC, EEE, and EMB, respectively (adjusted P < 0.01, log2FC > 2). Key regulators of trophoblast differentiation (Cdx2, Hand1, Nfat5, Ppard, Elf5, Pou3f1) were dysregulated in the EPC and EEE. Interestingly, markers of parietal TGCs (Prl3d1 and Prl4a1) were down-regulated in Med12 null EPC, seemingly in contrast to the increased TGC population observed at E9.5. Considering that other prolactin receptors used as markers of both TGC and EPC were also dysregulated in EPC and EEE (Prl2a1, Prl2b1, Prl2c), the molecular identity of the TGCs observed in E9.5 Med12 null implants may be compromised. Finally, genes related to chromatin organisation (Brd1, Ezh1, Hdac5, Kdm2a, Kmt2c, Kmt2e) were consistently up-regulated in all E7.5 tissues from Med12 null embryos, suggesting wide-spread epigenetic perturbation. Considering that Med12 is known to interact with chromatin modifiers (e.g. G9a and EP300), we hypothesise that loss of Med12 disrupts the embryonic epigenome, leading to downstream defects in lineage specification and eventual embryonic lethality.