67 CORRELATION OF ARCHITECTURAL AND FUNCTIONAL REPROGRAMMING OF NUCLEI DURING EMBRYONIC GENOME ACTIVATION OF EARLY BOVINE EMBRYOS GENERATED BY IVF AND SCNT

Abstract

Development of mammalian pre-implantation embryos provides an excellent model to explore interactions of nuclear organisation and nuclear functions. Based on light optical sectioning with confocal laser scanning microscopy and structured illumination microscopy, we performed a quantitative three-dimensional image analysis of nuclei in early bovine embryos generated by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) of bovine fibroblast nuclei. The same sequence of changes was observed in nuclei of both IVF and SCNT embryos during embryonic genome activation (EGA) is that typically achieved in embryos between 8 and 16 cells. In both pre-EGA IVF and SCNT embryos, chromosome territories (CT) were assembled as spatially distinct entities at the nuclear periphery, whereas the nuclear interior was typically occupied by a mostly chromatin free lacuna enriched with splicing factors. Detection of H3K4m3 demonstrates the presence of transcriptionally competent chromatin before EGA, which was correlated with large-scale movements of CT into the nuclear interior and a several-fold decrease of nuclear volumes. Post-EGA nuclei are characterised by a conventional nuclear architecture with chromatin distributed throughout the nuclear space, heterochromatin enriched with histone markers for transcriptionally silent chromatin beneath the nuclear lamina and around nucleoli, as well as heterochromatin clusters and chromocenters throughout the nuclear interior. Pre- and post-EGA nuclei were recorded with the superior resolution of structured illumination microscopy to allow a quantitative analysis of the nuclear topography of H3K4me3 and RNAP II signals. These signals were highly significantly enriched in the perichromatin region (PR) surrounding the compact, transcriptionally silent interior of megabase-sized chromatin domains, which form the basic structural units of CT. The PR is in direct contact with interchromatin compartment (IC) channels starting at nuclear pores, permeating the nuclear space and harboring nuclear bodies in IC lacunas. Our findings support a model for the functional nuclear architecture based on spatially distinct, but co-aligned three-dimensional networks of an active and an inactive nuclear compartment. The active nuclear compartment is built up from the structurally and functionally interacting IC and PR, whereas the inactive nuclear compartment consists of the compact, transcriptionally silent core of chromatin domain clusters.

This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).