Funktion von OCT4 während der Entwicklung boviner Embryonen vor der Implantation
Zusammenfassung der Projektergebnisse
Before implantation, the mammalian embryo undergoes two lineage specifications, where differentiated cells separate, while a portion of the embryonic cells maintain a pluripotent state. First, differentiation of the trophectoderm (TE) leading to the formation of the blastocyst with an inner cell mass (ICM) occurs and second, development of the primitive endoderm (PE) and the pluripotent epiblast (EPI) within the ICM. The fundamental mechanisms regulating these events have been studied extensively in the mouse, while advances in genome editing have enabled researchers to study the specific function of genes during preimplantation development in alternative model organisms. Given the substantial differences in the regulation of lineage differentiation and the maintenance of pluripotency between mouse and other mammalian species, this progress harbors the prospect of a deeper understanding of preimplantation development, also in human. The transcription factor OCT4/POU5F1 is at the center of a pluripotency regulatory network and regulates the maintenance of pluripotency as well as differentiation events during the second lineage differentiation. Because in vitro embryo production techniques are highly advanced in bovine, this species offers great opportunities as a model for preimplantation development. Therefore, this project was designed to study the role of OCT4/POU5F1 in the early bovine embryo using a reverse genetics approach. Using CRISPR/Cas9, a method to edit the genome, we disrupted the function of OCT4/POU5F1 in somatic fibroblast cells and used these cells for somatic cell nuclear transfer (cloning) to produce embryos with non-functional OCT4/POU5F1. We analyzed the effect of loss of OCT4/POU5F1 on embryonic development using fluorescent staining of lineage-specific markers, i.e., identification of cells in the TE, ICM, EPI and PE, and by transcriptome analyses using RNA sequencing. We found, that in contrast to mouse but similar to human development, OCT4/POU5F1 is essential for the normal expression of NANOG, a marker of the EPI lineage and directly linked to the maintenance of pluripotency. We also observed failure to develop the PE in embryos lacking OCT4/POU5F1, therefore in bovine as in mouse, this transcription factor is required to induce the second lineage differentiation during preimplantation development. To exclude, that our observations were biased by the cloning procedure or the in vitro culture of embryos, we first investigated the first and the second lineage differentiation in wildtype embryos that developed completely in vivo, which we obtained by flushing the uterus non-surgically. To circumvent the cloning procedure, we repeated the experiments by disrupting OCT4/POU5F1 directly in fertilized oocytes. Additionally, we transferred cloned embryos to recipient cows to exclude effects of a uterine environment. The effects of loss of OCT4/POU5F1 that we had observed in cloned embryos cultured in vitro were all confirmed, showing that our experimental approach provides a general strategy for studying the roles of specific genes during early embryonic development. The similarities in the function of OCT4/POU5F1 between bovine and human embryos show, that bovine embryos are an interesting model to study early human development.
Projektbezogene Publikationen (Auswahl)
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Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies. Cell Cycle, 17(14), 1688-1695.
Simmet, Kilian; Zakhartchenko, Valeri & Wolf, Eckhard
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OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proceedings of the National Academy of Sciences, 115(11), 2770-2775.
Simmet, Kilian; Zakhartchenko, Valeri; Philippou-Massier, Julia; Blum, Helmut; Klymiuk, Nikolai & Wolf, Eckhard
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Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos. Scientific Reports, 8(1).
Lavagi, Ilaria; Krebs, Stefan; Simmet, Kilian; Beck, Andrea; Zakhartchenko, Valeri; Wolf, Eckhard & Blum, Helmut
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Targeting aGal epitopes for multi-species embryo immunosurgery. Reproduction, Fertility and Development, 31(4), 820-826.
Kurome, Mayuko; Baehr, Andrea; Simmet, Kilian; Jemiller, Eva-Maria; Egerer, Stefanie; Dahlhoff, Maik; Zakhartchenko, Valeri; Nagashima, Hiroshi; Klymiuk, Nikolai; Kessler, Barbara & Wolf, Eckhard
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Gene Editing in Primary Cells of Cattle and Pig. Methods in Molecular Biology, 271-289. Springer New York.
Vochozkova, Petra; Simmet, Kilian; Jemiller, Eva-Maria; Wünsch, Annegret & Klymiuk, Nikolai
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Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How. International Journal of Molecular Sciences, 22(1), 236.
Simmet, Kilian; Wolf, Eckhard & Zakhartchenko, Valeri
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A New Toolbox in Experimental Embryology—Alternative Model Organisms for Studying Preimplantation Development. Journal of Developmental Biology, 9(2), 15.
Springer, Claudia; Wolf, Eckhard & Simmet, Kilian
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Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG. Cells, 10(9), 2232.
Springer, Claudia; Zakhartchenko, Valeri; Wolf, Eckhard & Simmet, Kilian
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OCT4/POU5F1 is indispensable for the lineage differentiation of the inner cell mass in bovine embryos. The FASEB Journal, 36(6).
Simmet, Kilian; Kurome, Mayuko; Zakhartchenko, Valeri; Reichenbach, Horst‐Dieter; Springer, Claudia; Bähr, Andrea; Blum, Helmut; Philippou‐Massier, Julia & Wolf, Eckhard
