title: Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos.
abstract: Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.
authors.affiliation: a Division of Anthropology and Human Genetics ; Biocenter; LMU Munich ; Munich , Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Popken
authors.forename: Alessandro
authors.initials: A
authors.lastname: Brero
authors.forename: Daniela
authors.initials: D
authors.lastname: Koehler
authors.forename: Volker J
authors.initials: VJ
authors.lastname: Schmid
authors.forename: Axel
authors.initials: A
authors.lastname: Strauss
authors.forename: Annegret
authors.initials: A
authors.lastname: Wuensch
authors.forename: Tuna
authors.initials: T
authors.lastname: Guengoer
authors.forename: Alexander
authors.initials: A
authors.lastname: Graf
authors.forename: Stefan
authors.initials: S
authors.lastname: Krebs
authors.forename: Helmut
authors.initials: H
authors.lastname: Blum
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: Thomas
authors.initials: T
authors.lastname: Cremer
mesh_terms: D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics*; D002843:Chromatin / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D019976:Cloning, Organism; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D011495:Histone-Lysine N-Methyltransferase; D012319:RNA Polymerase II
keywords: 3D-CLSM, 3-dimensional confocal laser scanning microscopy; 3D-SIM, 3-dimensional structured illumination microscopy; B23, nucleophosmin B23; BTA, Bos taurus; CDC, chromatin domain cluster; CT, chromosome territory; EM, electron microscopy; ENC, embryonic nuclei with conventional nuclear architecture; ENP, embryonic nuclei with peripheral CT distribution; H3K4me3; H3K4me3, histone H3 with tri-methylated lysine 4; H3K9me3; H3K9me3, histone H3 with tri-methylated lysine 9; H3S10p, histone H3 with phosphorylated serine 10; IC, interchromatin compartment; IVF, in vitro fertilization; MCB, major chromatin body; PR, perichromatin region; RNA polymerase II; RNA polymerase II-S2p, RNA polymerase II with phosphorylated serine 2 of its CTD domain; RNA polymerase II-S5p, RNA polymerase II with phosphorylated serine 5 of its CTD domain; SC-35, splicing factor SC-35; SCNT, somatic cell nuclear transfer.; bovine preimplantation development; chromatin domain; chromosome territory; embryonic genome activation; in vitro fertilization (IVF); interchromatin compartment; major EGA, major embryonic genome activation; somatic cell nuclear transfer (SCNT)