Temporal variability and cell mechanics control robustness in mammalian embryogenesis

Abstract

How living systems achieve precision in form and function despite their intrinsic stochasticity is a fundamental yet open question in biology. Here, we establish a quantitative morphomap of pre-implantation embryogenesis in mouse, rabbit and monkey embryos, which reveals that although blastomere divisions desynchronise passively without compensation, 8-cell embryos still display robust 3D structure. Using topological analysis and genetic perturbations in mouse, we show that embryos progressively change their cellular connectivity to a preferred topology, which can be predicted by a simple physical model where noise and actomyosin-driven compaction facilitate topological transitions lowering surface energy. This favours the most compact embryo packing at the 8- and 16-cell stage, thus promoting higher number of inner cells. Impairing mitotic desynchronisation reduces embryo packing compactness and generates significantly more cell mis-allocation and a lower proportion of inner-cell-mass-fated cells, suggesting that stochasticity in division timing contributes to achieving robust patterning and morphogenesis.

Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

“…Given that the physiological time window for ICM sorting in the developing embryo is approximately 12 hours, with the process commencing around E3.75 and completing by E4.5, the viability and the maintained EPI/PrE proportion of double-size mouse embryos (Saiz et al 2016(Saiz et al , 2020Tarkowski 1961) suggest additional mechanisms that facilitate the process. Thus, while our simulation shows that differential fluid-cell tension may explain the timely sorting of EPI/PrE cells in normal-size ICMs, this mechanism is not robust to variations that mouse embryos have in developmental progression and embryo size (Fabrèges et al 2023;Ichikawa et al 2022). This points to additional mechanisms contributing to EPI/PrE sorting within the ICM that ensure robust patterning of the mouse blastocyst.…”

“…The total cell number in the ICM was unchanged after immunosurgery (Figure S1A), and those in the ICMs isolated at E3.5 and E4.5, or after 24-hour culture from E3.5, were comparable to ICM cell numbers in E3.5 and E4.5 whole blastocysts, respectively (Figure 1B). We live-imaged the isolated ICMs using a fluorescent reporter of PrE fate, Pdgfra H2B-GFP , (Hamilton et al 2003;Plusa et al 2008) combined with a ubiquitous H2B-mCherry reporter (Abe et al 2011) (Figure 1C, and Video S1), and quantitatively analysed the dynamics of cell sorting, using a custom, semi-automated nuclear detection and tracking pipeline (Fabrèges et al 2023) (Figure 1D). To quantify ICM segregation, we define the sorting score as the extent of overlap between EPI and PrE spatial domains (Figure S1B), which describes both live and immunostained ICMs (Figure 1E, Figure S1C).…”

“…For ICMs isolated from mouse blastocysts, nuclear detection and tracking of cell centres was performed with a semi-automatic analysis pipeline developed by (Fabrèges et al 2023). In brief, centres of all nuclei were detected from time-lapse images using a Difference of Gaussians algorithm (Faure et al 2016) (DoG) using the nuclear fluorescence signal.…”

“…Using the highperformance computing cluster at EMBL, the best parameters for the DoG algorithm were found by a grid-search procedure which explored thousands of different configuration parameters simultaneously. The best output was manually curated and used for cell tracking with a nearest neighbour algorithm (Fabrèges et al 2023). Manual curation from the E3.5 to E4.0 stage of the ICM was performed and validated using the software Mov-IT by one operator.…”

“…Thus far, cell fate specification and spatial segregation have been studied independently, and an integrative view of ICM patterning is lacking. Specifically, it remains unclear whether EPI and PrE cells exhibit distinct dynamic movements in the ICM, and if so, what drives them, and how robust patterning is ensured in mouse embryos exhibiting spatio-temporal developmental variabilities, resulting in inevitable variability in embryo size and geometry (Dietrich and Hiiragi 2007;Fabrèges et al 2023;Ohnishi et al 2014;Plusa et al 2008). This is largely due to the technical challenge of analysing cellular dynamics in the presence of the rapidly changing geometry due to the expanding and collapsing fluid cavity.…”