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A systems-level understanding of the novel principle in early mammalian development

Final Report Summary - MAMMALIANDEVELOPMENT (A systems-level understanding of the novel principle in early mammalian development)

A fundamental question in biology is the mechanism by which the embryonic asymmetry is established during development. In contrast to many organisms in which embryonic development is driven by determinants localized asymmetrically in the egg, mammalian eggs lack the polarity and thus symmetry has to be broken during early embryogenesis. This symmetry breaking process in mammalian embryos result in formation of the blastocyst, composed of the inner cell mass (ICM) that will form the embryo proper, and a surrounding one-cell epithelial layer of the trophectoderm (TE) that will form extra-embryonic tissues. Despite its importance for understanding mammalian development and for stem cell research, the molecular mechanism of blastocyst patterning has long been elusive.

Recent studies including our own (Motosugi et al. 2005; Dietrich and Hiiragi 2007) revealed unexpectedly high dynamicity, stochasticity and cell-to-cell molecular heterogeneity during early embryogenesis. Collectively an attractive hypothesis is that early mammalian embryo may be a self-organization system patterning through stochastic processes in a particular structural context (Wennekamp et al. 2013). These features suggest that, in order to fully understand the mechanisms of early mammalian development, it will be essential to address how the diverse inputs acting on individual cells are integrated in the embryo at the systems level.

This project is thus aimed at establishing tools and multi-disciplinary strategies as a basis for the systems-level understanding of early mammalian development. Specifically we developed fluorescence-based gene-trap mouse lines that visualize molecular dynamics during embryonic patterning (Dietrich et al. manuscript in preparation), and characterised gene expression profile of every single cells of the ICM during blastocyst development (Ohnishi et al. 2014).

Our fluorescent gene-trap screen successfully generated reporter mice that express Venus specifically in one of the first lineages emerging in the blastocyst. We have also successfully established a high-resolution 4D live-imaging system suitable for lineage tracking and quantitative gene-expression and cell-position analyses, and this allowed us to generate a comprehensive lineage map of mouse pre-implantation development. The systematic analysis of the pattern of lineage segregation revealed that, contrary to the available models, the timing and mechanism of lineage specification are distinct between the TE and the ICM. The expression of a TE-specific lineage marker is up-regulated upon asymmetric divisions, whereas the ICM marker is not, and a consistent asymmetry in the ICM gene expression is evident only at the late blastocyst stage. This study thus provides a framework for systems-level analysis of embryogenesis that involves high dynamicity and stochasticity.

In a complementary approach, we characterised the expression profile of individual ICM cells during blastocyst development, and established a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the ICM. Clustering analysis demonstrated that initially the ICM cells are equivalent. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a new model in which stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.

These two achievements will form a solid basis for systems-level understanding of early mammalian development.

The single-cell microarray data and its analysis code have been deposited to the ArrayExpress database with the accession number E-MTAB-1681. Venus-trap mouse lines are deposited to RIKEN BioResource Centre and will be made available to the scientific community upon publication.