Investigating the Role of Hippo Signaling Effector Protein YAP1 in the First Lineage Decision in Cattle Embryos

In vitro embryo production (IVP) has provided insights into early embryogenesis in the murine model, however our current understanding of embryogenesis in the bovine model is less complete. A crucial event in mammalian embryonic development is the first lineage specification, where the trophoblast (TB) and the inner cell mass (ICM) are formed. The formation of these distinct cellular lineages is governed by various cell signaling pathways, foremost among which is the Hippo signaling pathway. The Yes-associated protein 1 (YAP1) is a key effector of Hippo signaling and has been shown to be involved in the regulation of mouse cellular lineage fates. The manipulation of this molecular pathway will provide information on whether Hippo pathway-dependent or Hippo pathway-independent mechanisms are involved in the regulation of YAP1 localization and first lineage specification in cattle.

Currently, there is limited research regarding the role of Hippo signaling in the establishment of polarity in the bovine embryo. We found that upon RHOA inhibition via C3-transferase, polarity factors EZRIN and AMOT exhibit reduced expression at the apical domain. Although it was not determined whether impaired embryo compaction and polarization was associated with YAP1 activity, it is evident that RHOA signaling is required for normal bovine compaction.

This research illustrated that RHOA facilitates YAP1 intracellular distribution in bovine embryos as RHOA inhibition activates the Hippo signaling pathway and shuts down YAP1 activity. In contrast, LATS1/2 inhibition via Lats-ln-1 resulted in unexpected YAP1 expression, hence the exact involvement of LATS1/2 in YAP1 nuclear localisation remains unclear. Furthermore, we concluded that RHOA and thereby YAP1 activity is involved in blastocyst formation as inhibition of RHOA may impair cell properties required for development beyond the morula stage.

Bovine embryos have a longer pre-implantation period than murine embryos, emphasizing that a complex network of mechanisms are involved in the establishment of the first lineages. In this thesis, RHOA inhibition was not linked to the second lineage determination and its role in the first lineage commitment was negligible. We showed that YAP1 modulation via LATS inhibition may impact the localisation and expression of first lineage markers. However, misexpression of 2 lineage markers in LATS1/2 inhibited blastocysts suggests that Lats-ln-1 may have a deleterious effect on bovine embryos.

In summary, we proved that Hippo signaling is involved in cell polarity and blastocyst establishment in the bovine embryo. Due to the complexity of bovine blastocyst formation, it is apparent that an extensive network of effectors and mechanistic pathways must also be involved in lineage determination independent of the Hippo signaling pathway.