Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

Abstract

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

Fig. 1.

Transcriptome changes during human preimplantation development. (A) Principal component analysis showing that samples at the same stage of development cluster closer together than they do to any other sample. (B) Hierarchical sample classifications of normalized transcriptional profiling of differentially expressed genes. (C) GenePattern K-means analysis identifies ten clusters (1 to 10) of transcripts that change their expression between the MII oocyte and the 10-cell stage. ESC, embryonic stem cell.

Fig. 2.

Pairwise changes in the transcriptome during human preimplantation development. Linear regression and pairwise comparison by Student's t-test with Benjamini correction analysis comparing consecutive developmental stages. The overlap between the stage represented on the x-axis (blue) and that represented on the y-axis (red) is in black. OOCYTE, MII oocytes; ESC, embryonic stem cell.

Fig. 3.

Human embryonic genome activation and pluripotency program initiation. Quantitative real-time PCR analysis of (A) genes that are actively transcribed from the embryonic genome starting at the 2-cell (RBBP6, FAM90A1, RFPL4B), 4-cell (L1TD1) and 6- to 8-cell (ZSCAN4, H2AFZ) stage, (B) genes involved in the initiation of the pluripotency program (POU5F1, TDGF1, NANOG, SOX2 and KLF4) and (C) maternally inherited mRNAs (FIGLA, ZP1). Blue bars, control culture conditions; red bars, α-amanitin-supplemented culture medium. Units on the y-axis indicate fold changes of expression as compared with MII (unit of expression). Error bars indicate s.e.

Fig. 4.

Pluripotency matrix across preimplantation human development. The expression of the main pluripotency-related genes at different stages of development. Red indicates high expression, green indicates low expression. White indicates genes not present on the microarray. The size of the square reflects the relative importance of the gene for the acquisition or maintenance of pluripotency. (A) MII oocytes, (B) 2-cell embryos, (C) 4-cell embryos, (D) 6-cell embryos, (E) 8- to 10-cell embryos, (F) morula stage embryos, (G) blastocyst stage embryos, (H) early passage human ESCs.