108 Single-Cell RNA Sequencing Reveals Blastomere Heterogeneity and Early Lineage Specification Events in Bovine Embryos During Major Embryonic Genome Activation

Abstract

During early embryonic stages, gene products generated by the embryo acquire control over embryonic development. At the 8- to 16-cell stage, major embryonic genome activation (EGA) occurs in bovine embryos. Morphological observations, such as size of blastomeres and distribution of microvilli, suggest heterogeneity of individual cells already at this developmental stage. To study this heterogeneity on the transcriptome level, we performed single-cell RNA sequencing (scRNA-seq) of 161 blastomeres from 14 in vitro-produced bovine embryos at Day 2 and Day 3 post-fertilization. After removing the zona pellucida, blastomeres were mechanically separated in Ca²⁺- and Mg²⁺-free PBS, individually collected, and lysed. Complementary DNA libraries were prepared by the single cell RNA-barcoding and sequencing (SCRB-Seq) protocol. Exogenous RNA was added for quality control and cell specific barcodes and unique molecular identifiers (UMI) were used to enable pooling of libraries and to exclude PCR duplicates, respectively. After sequencing (Illumina HiSEqn 1500; 50 nt reads; Illumina Inc., San Diego, CA, USA), UMI were counted with the published Drop-seq pipeline (45,000 UMI on average per library) and cells with UMI count <2.000 were removed. Data were normalized based on UMI and non-supervised clustering analyses of single-cell data were performed (SC3 and M3Drop R packages). The transcriptome profiles of all individual cells were assigned to 6 clusters with specific sets of genes. Sorting cells according to their transcriptome profiles by the CellTree R package (Bioconductor; https://bioconductor.org/packages/release/bioc/html/cellTree.html) resulted in a linear pseudo-timeline. Furthermore, this tool identified 6 groups of genes (topics). Each of them showed an over-representation of distinct Gene Ontology (GO) terms; topic 1, “translation” and “cell division”; topic 2, GO terms involved in translation, RNA splicing and cell division; topic 3, “translation”; topic 4, “ATP synthesis coupled proton transport”; topic 5, “mitochondrial translational elongation”; topic 6, “organic hydroxyl compound transport”. Moreover, increased expression of PCDH10 (protocadherin 10) was observed in the biologically pseudo-ordered more advanced blastomeres. This gene is known to be predominantly expressed in the inner cell mass (ICM) at the blastocyst stage, suggesting that these cells might become ICM. In summary, our study reveals developmental heterogeneity and hints to early lineage specification events in bovine embryos at the time of major EGA.