Role of yes-associated protein 1, angiomotin, and mitogen-activated kinase kinase 1/2 in development of the bovine blastocyst†

Abstract

The morula-stage embryo is transformed into a blastocyst composed of epiblast, hypoblast, and trophectoderm (TE) through mechanisms that, in the mouse, involve the Hippo signaling and mitogen-activated kinase (MAPK) pathways. Using the cow as an additional model, we tested the hypotheses that TE and hypoblast differentiation were regulated by the Hippo pathway regulators, yes-associated protein 1 (YAP1) and angiomotin (AMOT), and MAPK kinase 1/2 (MAPK1/2). The presence of YAP1 and CDX2 in the nucleus and cytoplasm of MII oocytes and embryos was evaluated by immunofluorescence labeling. For both molecules, localization changed from cytoplasmic to nuclear as development advanced. Inhibition of YAP1 activity, either by verteporfin or a YAP1 targeting GapmeR, reduced the percent of zygotes that became blastocysts, the proportion of blastocysts that hatched and numbers of CDX2+ cells in blastocysts. Moreover, the YAP1-targeting GapmeR altered expression of 15 of 91 genes examined in the day 7.5 blastocyst. Treatment of embryos with an AMOT targeting GapmeR did not affect blastocyst development or hatching but altered expression of 16 of 91 genes examined at day 7.5 and reduced the number of CDX2+ nuclei and YAP1+ nuclei in blastocysts at day 8.5 of development. Inhibition of MAPK1/2 with PD0325901 did not affect blastocyst development but increased the number of epiblast cells. Results indicate a role for YAP1 and AMOT in function of TE in the bovine blastocyst. YAP1 can also affect function of the epiblast and hypoblast, and MAPK signaling is important for inner cell mass differentiation by reducing epiblast numbers.

Yes-associated protein (YAP) is required in maintaining normal ovarian follicle development and function

“…Additionally, as seen in other species, disruption of hippo core elements leads to ovarian genetic abnormalities. In the bovine, nuclear YAP regulates hypoblast embryonic differentiation and inhibition of YAP activity reduces the percent of zygotes that became blastocysts [26]. To our knowledge, this is the first study to examine the role of YAP in bovine granulosa cells.…”

Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

“…YAP1 is expressed in the nuclei of most cells from day 5 onwards but not co-expressed with NANOG in later stages [13]. Knockdown of YAP1 or inhibition of the interaction between TEAD4 and YAP1 using verteporfin both result in a decreased number of CDX2 positive cells, indicating a role in TE differentiation [13]. But while in mouse knockout of Tead4 results in the embryo's inability to form a blastocyst and is lethal before implantation [14], knockdown of TEAD4 transcripts in bovine had no effect on the development of zygotes until blastocyst stage, while effects on the expression levels of TE specific genes remain controversial [12,15].…”

“…Nevertheless, the role of FGF4 is not analog to the situation in the mouse: inhibition of MAPK signaling only partially blocks GATA6 expression and increases the number of NANOG positive cells; inhibition of the FGF receptor has no effect on the composition of the ICM in the bovine embryo, while addition of FGF4 leads to GATA6 expression in all ICM cells. Therefore, FGF signaling is not essential for expression of GATA6 and the effect of exogenous FGF4 on GATA6 expression is indirect in bovine embryos [13,22]. Interestingly, in rabbit embryos exogenous FGF4 also induces PE fate in all cells of the ICM and inhibition of FGF/MAPK signaling prevents cells from acquiring a PE fate.…”

“…In bovine, polarization of outside cells and expression of TEAD4 is first observed at the 16-cell stage [11,12]. YAP1 is expressed in the nuclei of most cells from day 5 onwards but not co-expressed with NANOG in later stages [13]. Knockdown of YAP1 or inhibition of the interaction between TEAD4 and YAP1 using verteporfin both result in a decreased number of CDX2 positive cells, indicating a role in TE differentiation [13].…”

Role of ROCK signaling in formation of the trophectoderm of the bovine preimplantation embryo

“…Embryos were evaluated for formation of the blastocoele cavity at day 7.5 post‐insemination. Some blastocysts were selected randomly at this time without respect to morphological status (early, expanding, hatching or hatched) and used for analysis of immunolocalization of YAP1 and CDX2 ( n = 27 blastocysts for vehicle and n = 33 blastocysts for Y‐27632) using procedures similar to those described earlier (Negrón‐Pérez & Hansen, ). Cells whose nuclei were positive for CDX2 were considered TE while cells whose nuclei were negative for CDX2 were considered ICM (Denicol et al, ).…”