Search

The early embryo is the natural prototype for the acquisition of totipotency, which is the potential of a cell to produce a whole organism. Generation of a totipotent embryo involves chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications. Understanding embryonic chromatin architecture and how this is related to the epigenome and transcriptome will provide invaluable insights into cell fate decisions. Recently emerging low-input genomic assays allow the exploration of regulatory networks in the sparsely available mammalian embryo. Thus, the field of developmental biology is transitioning from microscopy to genome-wide chromatin descriptions. Ultimately, the prototype becomes a unique model for studying fundamental principles of development, epigenetic reprogramming, and cellular plasticity. In this review, we discuss chromatin reprogramming in the early mouse embryo, focusing on DNA methylation, chromatin accessibility, and higher-order chromatin structure. [ABSTRACT FROM AUTHOR]

The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females is maintained without detectable turnover and cohesin is therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levels decline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using β-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.