Computational analysis of single-cell transcriptomics data elucidates the stabilization of Oct4 expression in the E3.25 mouse preimplantation embryo

Daniela Gerovska,Marcos J Araúzo-Bravo

doi:10.1038/s41598-019-45438-y

Abstract

Our computational analysis focuses on the 32- to 64-cell mouse embryo transition, Embryonic day (E3.25), whose study in literature is concentrated mainly on the search for an early onset of the second cell-fate decision, the specification of the inner cell mass (ICM) to primitive endoderm (PE) and epiblast (EPI). We analysed single-cell (sc) microarray transcriptomics data from E3.25 using Hierarchical Optimal k-Means (HOkM) clustering, and identified two groups of ICM cells: a group of cells from embryos with less than 34 cells (E3.25-LNCs), and another group of cells from embryos with more than 33 cells (E3.25-HNCs), corresponding to two developmental stages. Although we found massive underlying heterogeneity in the ICM cells at E3.25-HNC with over 3,800 genes with transcriptomics bifurcation, many of which are PE and EPI markers, we showed that the E3.25-HNCs are neither PE nor EPI. Importantly, analysing the differently expressed genes between the E3.25-LNCs and E3.25-HNCs, we uncovered a non-autonomous mechanism, based on a minimal number of four inner-cell contacts in the ICM, which activates Oct4 in the preimplantation embryo. Oct4 is highly expressed but unstable at E3.25-LNC, and stabilizes at high level at E3.25-HNC, with Bsg highly expressed, and the chromatin remodelling program initialised to establish an early naïve pluripotent state. Our results indicate that the pluripotent state we found to exist in the ICM at E3.25-HNC is the in vivo counterpart of a new, very early pluripotent state. We compared the transcriptomics profile of this in vivo E3.25-HNC pluripotent state, together with the profiles of E3.25-LNC, E3.5 EPI and E4.5 EPI cells, with the profiles of all embryonic stem cells (ESCs) available in the GEO database from the same platform (over 600 microarrays). The shortest distance between the set of inner cells (E3.25, E3.5 and E4.5) and the ESCs is between the E3.25-HNC cells and 2i + LIF ESCs; thus, the developmental transition from 33 to 34 cells decreases dramatically the distance with the naïve ground state of the 2i + LIF ESCs. We validated the E3.25 events through analysis of scRNA-seq data from early and late 32-cell ICM cells.

Highlights

The mouse preimplantation development begins with the division of the 1-cell zygote to progressively smaller cells, blastomeres, forming the morula, which at the 8-cell stage compacts
Focusing on the E3.25-E3.5 stages and using only the inner cell mass (ICM) sc data from Ohnishi et al.[3] (Fig. 1B), the Principal Component Analysis (PCA) revealed that E3.25 cells divide into two groups, one comprised mainly of cells from embryos with lower number of cells (LNCs), between 32 and 33, and cell 26 C41 IN from a 41-cell embryo, and another comprised of cells from embryos with higher number of cells (HNCs), between 34 and 50
We found that the HNC-h-differently expressed genes (DEGs) are lowly expressed in E3.25 Fgf4 knockouts (Fgf4-KO) and E4.5 Fgf4-KO cells, they are highly expressed in E3.5 Fgf4-KO, the transcriptomics profile of E3.5 Fgf4-KO resembles the profile of E3.25-HNCs

Summary

Introduction

The mouse preimplantation development begins with the division of the 1-cell zygote to progressively smaller cells, blastomeres, forming the morula, which at the 8-cell stage compacts. Ohnishi et al.[3] found bimodal expression of Fgf[4] within the E3.25 ICM cells, and suggested that as an early indication of future PE or EPI fate. We hypothesized that such bimodal expression of Fgf[4], among others, could be traced to earlier developmental stages and performed a bifurcation analysis on the sc data of Ohnishi et al.[3], spanning the time from E3.25, E3.5 to E4.5, while introducing an additional bifurcation point (between E3.25-LNC and E3.25-HNC) during E3.25, owing to the newly identified E3.25 sub-stages. We built a simple cell-packing model to explain the strong transcriptional changes between ICM cells from embryos with less than 34 cells and ICM cells from embryos with more than 33 cells

Methods

Results

Conclusion