Investigating the role of Hippo signalling pathway during human pre- and peri-implantation embryo development reveals novel insights in early embryo lineage segregation

Understanding the mechanisms underlying human embryo development and embryo implantation
failure is crucial, as 40-45% of the euploid embryos fail to develop to the blastocyst stage, and
25-30% of the formed blastocysts fail to implant. Therefore, we need fundamental insights in the
signalling pathways regulating embryo lineage segregation. Although we have raised the importance
of Hippo signalling pathway on embryo lineage segregation, major aspects such as the distinct roles
of canonical vs non-canonical Hippo signalling, and the role of Hippo signalling independent factor
(VGLL4), remain elusive. I hypothesize that canonical Hippo signalling is the master regulator of
lineage segregation, with VGLL4 regulating the transcriptional output of the pathway through
competitive interaction with the Hippo effectors. In this way Hippo signalling pathway controls the
segregation in the distinct lineages. I aim to functionally characterize canonical Hippo signalling and
the role VGLL4 via pharmacological inhibition of its core kinases and by gene targeting using CRISPRCas9 respectively. Effects will be examined on embryo morphology, development, kinetics and
expression of lineage specific markers/Hippo signalling associated genes by immunofluorescence
imaging and single-cell RNA sequencing. The ability of the embryos to attach and implant will be
investigated by utilizing state of the art endometrial gland organoids, which very closely recapitulate
the human endometrium.