O-072 The canonical Hippo signalling pathway controls the first lineage segregation in the human preimplantation embryo
Authors: Stamatiadis P , Regin M , De Munck N , Tournaye H , Sermon K , Van De Velde H 
Published: July 2024

Abstract

Study question
What is the role of the canonical Hippo signalling pathway during the first lineage segregation in the human preimplantation embryo?

Summary answer
Canonical Hippo signalling regulates the first lineage segregation by controlling the expression of trophectoderm (TE) and inner cell mass (ICM) associated genes.

What is known already
The Hippo signalling pathway is a conserved regulatory network that plays a critical role in the first lineage segregation in mouse, rat, bovine and human preimplantation embryos. This pathway consists of two distinct branches (canonical, non-canonical). Despite their shared role in lineage segregation, the respective functions of canonical and non-canonical Hippo signalling branches remain largely unexplored. Since these branches are regulated by distinct upstream mechanisms, they may have different roles in embryonic growth, function, and survival. Unravelling the distinct roles of these branches is essential for understanding the intricate mechanisms that govern early human embryonic development.

Study design, size, duration
This experimental study involved the functional characterization of the canonical Hippo signalling pathway by pharmacologically inhibiting the core kinase MST1/2. Eight-cell stage human embryos donated for research were warmed and incubated with or without the inhibitor, till completion of the first lineage segregation days post fertilization (dpf) 5. The control (n = 20) and treated embryos (n = 17) were evaluated based on embryonic development (kinetics, morphology), and expression of lineage-specific markers.

Participants/materials, setting, methods
The optimal concentration (1μΜ) of the highly selective XMU-MP-1 inhibitor targeting the MST1/2 core kinase of the canonical Hippo signalling pathway was established in titration experiments. Time-lapse imaging (Miri TL, Esco Medical) captured effects on embryonic development (kinetics, morphology). Changes in the main effectors of the Hippo signalling pathway (YAP1, p-YAP1 and WWTR1) and the expression of lineage-specific markers (ICM: NANOG, SOX2; TE: GATA3) were assessed through immunofluorescence imaging and confocal microscopy (LSM800, Zeiss).

Main results and the role of chance
For the first time, we provide a complete spatiotemporal analysis of the main effectors of the Hippo signalling pathway YAP1 and WWTR1. They are co-localised in the nuclei of outer cells in compacting (dpf3) and compacted (dpf4) embryos. In the dpf5 blastocyst, YAP1 and WWTR1 are detected in the nuclei of the TE cells. Phosphorylated effector p-YAP1, indicative of active Hippo signalling, was first detected in the inner cells of the compacted embryo (dpf4) and remained in the ICM of the dpf5 blastocyst, supporting the role of the Hippo signalling pathway in the first lineage segregation. Treatment of human embryos with XMU-MP-1 reduced p-YAP1 levels, confirming its specificity for MST1/2. Inhibition of the MST1/2 kinase had a significant effect on the developmental kinetics as treated embryos had lower blastocyst formation rates. Embryo morphology was significantly compromised as treated embryos formed ICMs with less cells compared to control embryos. Immunofluorescence analysis revealed that the XMU-MP-1 treatment led to the upregulation of YAP1 and upregulation of TE marker GATA3, while the ICM markers SOX2 and NANOG were downregulated. Overall, our data suggest that active Hippo signaling promotes ICM formation, while TE formation requires inactive Hippo signaling.

Limitations, reasons for caution
Caution is warranted due to potential non-specific effects of the XMU-MP-1 inhibitor, although this compound has been shown in other models to be very specific. Further studies should explore off-target impacts to ensure result reliability.

Wider implications of the findings
Understanding the signalling pathways that regulate human preimplantation embryo development is fundamental for understanding embryonic growth, function, and survival, and can also help improve IVF success rates by addressing deficiencies that lead to embryo developmental arrest.

Trial registration number
Not applicable