Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

Abstract

Recent technological advances such as single-cell RNAseq 1-3 and CRISPR-CAS9-mediated knock-out 4 have allowed an unprecedented access into processes orchestrating human preimplantation development 5 . However, the sequence of events which occur during human preimplantation development are still unknown. In particular, timing of first human lineage specification, the process by which the morula cells acquire a specific fate, remains elusive. Here, we present a human preimplantation development model based on transcriptomic pseudotime modelling of scRNAseq biologically validated by spatial information and precise time-lapse staging. In contrast to mouse, we show that trophectoderm (TE) / inner cell mass (ICM) lineage specification in human is only detectable at the transcriptomic level at the blastocyst stage, just prior to expansion. We validated the expression profile of novel markers enabling precise staging of human preimplantation embryos, such as IFI16 which highlights establishment of epiblast (EPI) and NR2F2 which appears at the transition from specified to mature TE. Strikingly, mature TE cells arise from the polar side, just after specification, supporting a model of polar TE cells driving TE

Generation of human induced trophoblast stem cells

“…We further clustered cells on the UMAP, which yielded 19 clusters of cells. In particular, this distinguished clusters associated with the development of the trophoblast lineage: early, medium and late pre-implantation TE, in line with our recent report (Meistermann et al, 2019b), 5 postimplantation trophoblast (TB #1 to TB #5), then pre-EVT, EVT, pre-ST and ST ( Figure 4A).…”

“…We finally analyzed hiTSC and hcTSC by immunofluorescence for the trophoblast markers NR2F2 and GATA2, that are expressed in the trophectoderm of human blastocysts (Meistermann et al, 2019a). NR2F2 and GATA2 were highly expressed and localized in nuclei of all cells.…”

“…The markers that are better associated with hTSC are found in the gene sets specific of clusters TB #1, TB #2 and TB #3. Among those gene lists, we identified markers that have been assessed by immunofluorescence in human embryos: NR2F2, CDX2, GATA3, KRT7, CCR7 (Deglincerti et al, 2016;Meistermann et al, 2019b;Niakan and Eggan, 2013;Petropoulos et al, 2016). Projection of the expression of those markers on the UMAP led us to propose that hTSC expressed genes associated with clusters TB #1 and TB #2.…”

“…Data analysis and transcriptomic modeling of human preimplantation development is detailed in Meistermann et al (Meistermann et al, 2019a).…”

“…The n_neighbors parameter was set to its maximum (6838), and min_dist = 0.2. A first cell clustering was done using Monocle3 (Qiu et al, 2017), then, preimplantation clusters were re-annotated using clusters found in Meistermann et al (Meistermann et al, 2019a). First, coordinates of centroids of these previous clusters were determined on the UMAP using Petropoulos dataset, then, cells were attributed to the cluster with the nearest centroid in term of Euclidean distance.…”