Mei2Mit

Mammalian oocytes are arrested in the second meiosis until fertilisation triggers completion of the meiotic cell cycle and initiation of the mitotic one: the meiotic-to-mitotic transition in one-cell embryos. The control of both meiosis and mitosis involves much-studied post-transcriptional mechanisms, including protein modification and removal, but mitosis is also regulated at the level of transcription. Although it seems likely that parallel transcriptional mechanisms operate in the meiotic-to-mitotic transition, very little is known about them in any species. Recent evidence from the applicants suggests that following fertilisation, transcription in the newly-formed one-cell embryo (immediate embryonic genome activation, iEGA) regulates the cell cycle and plays a pivotal role in healthy embryonic development. This proposal sets out to provide a mechanistic model accounting for how such transcription regulates the meiotic-to-mitotic transition in mouse one-cell embryos by unifying two core research themes: exit from second meiotic arrest, and the onset of embryonic transcription. Meiotic arrest is principally due to the cytostatic factor, endogenous meiotic inhibitor 2, Emi2. Oocytes artificially depleted of Emi2 autonomously complete meiosis and initiate abortive development: Emi2 thus pivotally coordinates meiotic exit, mitotic entry and developmental initiation. Emi2 protein levels precipitously decline immediately after fertilisation, but Emi2 transcript levels increase. Emi2 expression at high levels in transgenic mouse lines causes meiotic arrest, but lower levels result in parthenogenesis and tumour formation, cohering with recent evidence that EMI2 functions as a human oncogene. The second theme is integrated with the first, and relates to our recent demonstration that iEGA initiates within four hours of fertilisation. Gene regulatory networks upregulated in mouse and human one-cell embryos included cell-cycle terms and correctly predicted transcriptional regulators that, when blocked, precipitated cell-cycle arrest. We will pursue these findings via three Objectives. Objective 1 will identify in silico transcription regulator binding motifs in genes with candidate roles in the meiotic-to-mitotic transition: expression of corresponding mRNA and protein will be evaluated experimentally. Objective 2 will disrupt Emi2 and transcriptional regulators of Objective 1 in one-cell embryos and the effects on the embryonic cell cycle, development and gene expression (assessed by single-cell RNA sequencing and validated by qPCR) determined. Objective 3 will identify and dissect regulator associations with chromatin in oocytes and one-cell embryos and relate interactions to cell-cycle gene control. Outputs will allow us to build a mechanistic model of how the cell cycle is regulated at the onset of mammalian development. Transcriptional regulation in very early embryogenesis is unique and relatively unexplored, but promises new opportunities to illuminate processes in cancer and pluripotency, with which it shares parallels. The proposal capitalises on a long-standing and productive collaboration between embryologists and bioinformaticians in Bath and Cambridge. Cells cultured in vitro will be utilised where possible, but animal work is unavoidable in studies on early embryology. The proposal fits several priority areas, presenting novel ideas for an integrated understanding of health to reveal the rules of life (eg new roles for transcriptional cell-cycle regulation) through transformative technologies (eg novel embryo manipulation). It will have ramifications for multiple fields in which embryology is a component, including assisted reproduction, mitochondrial replacement, embryoid culture and nuclear transfer cloning. It is expected to yield high-impact publications and produce the first model of cell-cycle regulation by control of gene expression at the onset of mammalian development.