Inhibition of RHO–ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst

Abstract

Specification of the trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst correlates with cell position, as TE derives from outer cells whereas ICM from inner cells. Differences in position are reflected by cell polarization and Hippo signaling. Only in outer cells, the apical-basal cell polarity is established, and Hippo signaling is inhibited in such a manner that LATS1 and 2 (LATS1/2) kinases are prevented from phosphorylating YAP, a key transcriptional co-activator of the TE-specifying gene Cdx2. However, the molecular mechanisms that regulate these events are not fully understood. Here, we showed that inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling and disruption of apical-basal polarity. Embryos treated with ROCK inhibitor Y-27632 exhibited elevated expression of ICM marker NANOG and reduced expression of CDX2 at the blastocyst stage. Y-27632-treated embryos failed to accumulate YAP in the nucleus, although it was rescued by concomitant inhibition of LATS1/2. Segregation between apical and basal polarity regulators, namely PARD6B, PRKCZ, SCRIB, and LLGL1, was dampened by Y-27632 treatment, whereas some of the polarization events at the late 8-cell stage such as compaction and apical localization of p-ERM and tyrosinated tubulin occurred normally. Similar abnormalities of Hippo signaling and apical-basal polarization were also observed in embryos that were treated with RHO GTPases inhibitor. These results suggest that RHO-ROCK signaling plays an essential role in regulating Hippo signaling and cell polarization to enable proper specification of the ICM and TE lineages.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage

“…Incubation of 8-cell embryos in the presence of Y-27632, a specific inhibitor of ROCK1 and ROCK2, blocked blastocyst formation and resulted in a loss of position-dependent HIPPO signaling. These findings are largely consistent with those of a recent study which showed that RHO-ROCK signaling is required for trophectoderm specification (Kono et al, 2014). Specifically, the authors showed that ROCK controls position-dependent HIPPO signaling by repressing LATS activity in the outside cells.…”

“…To test whether apical cell polarity was altered in Y-27632-treated embryos, we evaluated the expression and localization of PARD6B. Consistent with a previous report (Kono et al, 2014), PARD6B was no longer largely confined to the apical region, but was localized to both the apical and basal region of the embryos (Fig. 6F).…”

“…Recently, it was reported that Rho-associated protein kinase (ROCK) regulates position-dependent HIPPO signaling through cell polarity and inhibition of LATS activity in the outside cells (Kono et al, 2014). Previously, we showed that Rock2 is a direct target gene of TFAP2C in early embryos and TS-like cells (Choi et al, 2012).…”

Integrin signalling regulates YAP and TAZ to control skin homeostasis

“…Mechanical force has been proposed to modulate signalling by both the apical Crb-Mer/Ex-Kibra-Sav system in Drosophila (Fletcher et al, 2015;Rauskolb et al, 2014) as well as the basal integrin-Src system in mammalian cell culture (reviewed in Humphrey et al, 2014;Lawson and Burridge, 2014). In the early mouse pre-implantation embryo, cortical tension is higher in outer cells than inner cells, leading to nuclear YAP in outer cells despite the presence of an apical domain (Anani et al, 2014;Kono et al, 2014;Nishioka et al, 2009). Consistently, reducing cortical tension with a ROCK inhibitor abolishes nuclear localisation of YAP in early mouse embryos (Anani et al, 2014;Kono et al, 2014;Nishioka et al, 2009).…”

Lineage specification in the mouse preimplantation embryo

“…This apical protein complex plays important roles in the formation of the blastomere apical domain (Plusa et al, 2005;Alarcon, 2010). Rho GTPase activity and microtubule dynamics are also required for initiating apical domain formation, but how they are linked to the apical protein complex is unknown (Clayton et al, 1999;Houliston et al, 1989;Kono et al, 2014).…”