106 STUDIES OF NUCLEAR ARCHITECTURE IN MAMMALIAN PRE-IMPLANTATION EMBRYOS AND EMBRYONIC STEM CELLS USING SUPER-RESOLUTION FLUORESCENCE MICROSCOPY

Abstract

Three-dimensional (3-D) super-resolution fluorescence microscopy has allowed major progress in studies of the functional nuclear organization (Markaki et al. 2010 Cold Spring Harb. Symp. Quant. Biol. 75, 475–492; Markaki et al. 2012 Bioessays 34, 412–426). We have exploited these new possibilities to explore nuclear organization at different stages of bovine pre-implantation development (4-cell, 8-cell, 16-cell, morula, and blastocyst stage). In particular, we studied the topography of RNA polymerase II and the distribution of transcriptionally competent and noncompetent chromatin using antibodies against H3K4me3 and H3K27me3, respectively. For comparison, we have started analyses of mouse pre-implantation embryos and embryonic stem cells as well. Our results support the chromosome territory-interchromatin compartment (CT-IC) model (Cremer and Cremer 2010 Cold Spring Harb. Perspect. Biol. 2, a003889; Cremer et al. 2012 In: Epigenetic Regulation and Epigenomics 451–483). In all cell types, the nuclear space is occupied by chromosome territories (CTs; Koehler et al. 2009 Exp. Cell Res. 315, 2053–2063), the interchromatin compartment (IC), and one or several nucleoli. The CTs are built up from interconnected, megabase-sized chromatin domains (CDs). These ~1-Mbp CDs may consist of a series of ~100-kbp CDs (Cremer et al. 2000 Crit. Rev. Eukaryot. Gene Expr. 10, 179–212), which globally form a compact chromatin core surrounded by a layer of decondensed chromatin, called the perichromatin region. Current evidence supports the hypothesis that the perichromatin region represents the nuclear compartment, where transcription, co-transcriptional splicing, DNA-replication, and DNA-repair take place (Rouquette et al. 2010 Int. Rev. Cell Mol. Biol. 282, 1–90). The IC provides a contiguous, crowded compartment, which starts with channels at nuclear pores and pervades the chromatin compartment both between and within CTs. Small-scale chromatin loops of the perichromatin region can protrude into the interior of IC channels allowing direct contacts between CDs in cis and trans. At other sites the IC expands to wider, chromatin-free lacunas with splicing speckles and nuclear bodies. This model is in line with a fractal higher-order chromatin arrangement at all levels from CTs, chromosome arms and bands to ~1 Mbp CDs organized as fractal globules (Mirny 2011 Chromosome Res. 19, 37–51). This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).