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Analysis of Blastomeres of Bovine Embryos During Genome Activation by Evaluation of Single-Cell RNA Sequencing Data

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Analysis of Blastomeres of Bovine Embryos During Genome Activation by Evaluation of Single-Cell RNA Sequencing Data Analysis of blastomeres of bovine embryos during genome activation by evaluation of single-cell RNA sequencing data von Ilaria Lavagi Inaugural-Dissertation zur Erlangung der Doktorwürde (Dr. rer. biol. vet.) der Tierärztlichen Fakultät der Ludwig-Maximilians- Universität München Analysis of blastomeres of bovine embryos during genome activation by evaluation of single-cell RNA sequencing data von Ilaria Lavagi aus Mailand München 2018 I Aus demVeterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie Arbeit angefertigt unter der Leitung von: Univ.-Prof. Dr. Eckhard Wolf Angefertigt im Laboratorium für Funktionale Genomanalyse (LAFUGA) Abteilung Genomics Genzentrum der Ludwig-Maximilians-Universität München Mentor: Dr. Helmut Blum II Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Reinhard K. Straubinger Berichterstatter: Univ.-Prof. Dr. Eckhard Wolf Korreferent/en: Priv. –Doz. Dr. Wolfram Petzl Tag der Promotion: 27. Juli 2018 III Contents Abbreviations ..................................................................................................................... 2 Introduction ........................................................................................................................ 3 Bovine Embryos as Model System ........................................................................................... 3 Cell Cleavage and Cell Stage ..................................................................................................... 3 Early Bovine Embryo Development ........................................................................................ 3 Embryonic Genome Activation (EGA) ................................................................................... 4 Epigenetic Modifications ............................................................................................................ 4 Lineage Specification at Early Stages of Embryo Development ........................................ 5 Aims of the Project ...................................................................................................................... 5 Publication .......................................................................................................................... 6 Abstract .......................................................................................................................................... 6 Introduction .................................................................................................................................. 7 Results ............................................................................................................................................. 7 Discussion ..................................................................................................................................... 20 Material and Methods ............................................................................................................... 24 Acknowledgements .................................................................................................................... 26 Author Contribution ................................................................................................................. 26 Supplementary Information .................................................................................................... 27 References ................................................................................................................................. 103 Discussion ....................................................................................................................... 107 The technology of Single-cell RNA sequencing ................................................................ 107 From Raw to Processed Data ............................................................................................... 107 Cell Population Characterization Based on Transcriptome Profiles .......................... 108 Pseudo-time of Blastomeres .................................................................................................. 109 Embryonic Genome Activation in each Blastomere ........................................................ 109 Genes inducing or Reflecting Cell Fate Decisions ............................................................ 110 The interesting finding of PCDH10 ..................................................................................... 111 Summary ......................................................................................................................... 112 Zusammenfassung ....................................................................................................... 113 References ...................................................................................................................... 114 1 Abbreviations rRNA Ribosomal RNA EGA Embryonic Genome Activation RNA-seq RNA-sequencing Dnmts DNA methyltransferases TE Trophectoderm ICM Inner Cell Mass PE Primitive Endoderm EP Pluripotent Epiblast qPCR Quantitative Real-Time PCR WISH Whole-Mount in situ Hybridization MET Maternal-to-Embryonic Transition scRNA Single-cell sequencing hpf Hours Post Fertilization SCRB-Seq Single-cell RNA Barcoding and Sequencing UMIs Unique Molecular Identifier(s) DATs Differentially Abundant Transcript(s) K Cluster SC3 Single-cell Consensus Clustering M3Drop Michaelis-Menten Modelling of Dropouts AUROC Area under the ROC Curve GO Gene Ontology LDA Latent Dirichlet Allocation IVM in vitro Maturation IVF in vitro Fertilization COC(s) Cumulus-Oocyte Complex(es) MPM Modified Parker Medium LH Luteinising Hormone FSH Follicle-Stimulating Hormone ECS Estrous Cow Serum SOF Synthetic Oviductal Fluid THP TALP-HEPES-PVP ZP Zona Pellucida FCS Foetal Calf Serum 2 Introduction Bovine Embryos as Model System Direct analysis of human embryos is restricted for ethical reasons and in many countries prohibited by specific regulations, such as the “German embryo protection law” in Germany. For this reason, embryos from animal species are used as models for studying early human development. The most widely used organism for this purpose is mouse, as mouse and human embryo implantation are very similar 1. However, as interactions between the embryo and the corpus luteum are very similar between human and bovine, the bovine animal represents a better model than mouse to study biochemical and maternal and paternal regulatory processes in human 1. Cell Cleavage and Cell Stage After fertilization the newly formed zygote starts dividing; cell cleavage is regulated by cell cycle. As blastomeres divide asynchronously from the four-cell stage onwards 2, embryos at the same developmental stage show different numbers of blastomeres, a fact that might be misinterpreted. The third cleavage cycle results into the eight-cell stage embryo, while the fourth cleavage results into 16-cells stage embryos. We refer to the eight-cell stage when the embryo is composed of 5-9 cells (Day 2 in vitro post fertilization). Similarly, we define the embryo at the 16-cell stage, when it is made of 13-17 cells (Day 3 in vitro post fertilization). Early Bovine Embryo Development After fertilization, the zygote undergoes multiple cell divisions. The embryo and its blastomeres change their morphology and function. These changes characterize the different developmental stages 3. In early embryonic stages, cell polarization is an important event in the transition from zygote to blastocyst, leading to different cell- types 4. Microvilli, membrane protrusions that increase the surface area, have been studied to model cell polarization. In the 9- to 15-cell bovine embryos two cell-types have been observed. Some cells are uniformly covered with long and short microvilli (non-polar), while others have a single pole of long microvilli (polar). From the 16- cell stage onwards, almost all bovine embryos possessed one or more blastomeres with a single pole of long microvilli (polar) 5. In addiction to cell polarization, the nucleologenesis and the onset of transcription play a crucial role in yielding different cell-types. The nucleolus is the site of ribosomal RNA (rRNA) synthesis; rRNA is an essential component of ribosomes, required for mRNA translation. At the four-cell stage only a fibrillar center is visible. This is the structure where the enzymes necessary for transcription are located. At the eight-cell stage, a dense fibrillar component is visible in addition to the fibrillar center. The dense fibrillar component carries the unprocessed rRNA transcripts 6. Interestingly, the starting point for morphogenetic events linked with the onset of transcription was identified at the eight-cell stage 7. Finally, at the 16-cell stage, the granular component is formed as 3 the third part of the nucleolus. Here the processed rRNAs are associated with proteins 6. Embryonic Genome Activation (EGA) At the time of embryo fertilization, maternal mRNAs and proteins support the development of the embryo. During early stages of embryonic development, the embryo starts gene expression and gradually degrades the maternal products, thus acquiring the control over its own embryonic development. This process is called Embryonic Genome Activation (EGA) and occurs in several waves 8. The timing of the major EGA is species-specific. In the mouse embryo it takes place at the
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