Histones are essential components of the nucleosomal structure of chromatin. Substitution by structural variants provides important epigenetic information that regulates gene expression. H2A.Z is an essential H2A variant, with H2a.z-null embryos dying around the time of implantation. To provide insights into its roles in the embryo, we examined localization of this histone across the stages of preimplantation embryo development.
Mouse embryos were collected at the 1-cell, 2-cell, 8-cell, morula and blastocyst stages. Embryos were fixed with 4% PFA, permeabilized with 0.5% Tween-20 and 0.5% TritonX-100, blocked in 30% donkey serum (2h at 22OC) and stained for H2A.Z via indirect immunofluorescence. Staining with sheep polyclonal anti-H2A.Z (1:300) at 4OC overnight and donkey anti-sheep-IgG FITC-labelled (1:250) (Sigma-Aldrich, F7634). Counterstaining of DNA with DAPI (Vectashield-H1200 with DAPI, 1.5 µg/ml) or PI (Sigma P-4170, 5- 10µg/ml).
Embryos were imaged by conventional (Eclipse 80i, Nikon, Japan) and confocal (Leica TCS SP5, Leica Microsystems Pty Ltd, Australia) microscopy. Dynamic patterns of staining were observed. Increased accumulation of H2A.Z staining occurred in both pronuclei during zygotic maturation, and a number of distinct staining foci appeared at the periphery of all nucleoli. In the 2-cell embryo (the time of embryonic genome activation) there is a marked loss of staining except for a single small but distinct staining focus in each nucleus. In the 8-cell embryo staining was intense, with bright staining surrounding of each presumptive nucleolus. By the morula stage more staining occurred within nucleoli and by the blastocyst stage this process is complete with most of the intense staining occurring within the presumptive nucleoli. In both morulae and blastocysts stage staining was similar in all cells of the embryo.The highly dynamic changes in H2A.Z localization and levels during key transitions in embryo development indicate possible differing roles for this essential histone variant during embryo development.