Figure 1. ROCK1/2 and nuclear YAP1 repress expression of SOX2.

(A) Experimental design: embryos were collected at E2.5 and treated with ROCK inhibitor Y-27632 (ROCKi) or DMSO (control) for 24 hr. (B–B’) Confocal images of apical (PARD6B) and basolateral (CDH1) membrane components in control and ROCKi-treated embryos. As expected, PARD6B and CDH1 are mislocalized to the entire cell membrane of all cells in ROCKi-treated embryos, demonstrating effective ROCK inhibition (n = number of embryos examined). (C–C’) In control embryos, SOX2 is detected only in inside cells, while in ROCKi-treated embryos, SOX2 is detected in inside and outside cells (arrowheads, outside cells; n = embryos). (D) Quantification of ectopic SOX2 detected in outside cells of control and ROCKi-treated embryos (p, student’s t-test, n = embryos). (E) SOX2 and CDX2 staining in outside cells of control and ROCKi-treated embryos. ROCK-inhibitor treatment leads to outside cells with mixed lineage marker expression (CDX2+/SOX2+). (F) Experimental design: embryos were collected at E1.5 and one of two blastomeres injected with mRNAs encoding YAP1^CA and GFP. Embryos were cultured for 72 hr, fixed, and then analyzed by immunofluorescence and confocal microscopy. (G) SOX2 is detected non-injected inside cells. SOX2 is not detected in YAP1^CA-overexpressing inside cells (arrowheads), n = embryos. (H) Across multiple embryos, all non-injected inside cells express SOX2, whereas the vast majority of YAP1^CA-injected inside cells fail to express SOX2.

Figure 1—figure supplement 1. Effect of ROCK1/2 inhibition on Cdx2 expression and effect Yap1^CA overexpression on YAP1 localization and phosphorylation.

(A) Confocal images of CDX2 and SOX2 in control embryos and embryos treated with ROCKi for 24 hr starting at E2.5.In control embryos, CDX2 is specific to outside cells and SOX2 is specific to inside cells (n = embryos). (A’) Treatment with ROCKi leads to ectopic SOX2 in outside cells which is often co-expressed with CDX2 (arrowheads, n = embryos). (B) YAP1^CA was injected into one of two blastomeres at the 2 cell stage and evaluated 48 hr later. (C) In non-injected cells, YAP1 is exclusively nuclear in outside cells while pYAP1 is exclusively cytoplasmic in inside cells. By contrast, YAP1 is detected in the nucleus of Yap1^CA-injected cells, regardless of their position, demonstrating that YAP1^CA is constitutively nuclear. Additionally, analysis of pYAP1 in YAP1^CA-injected cells shows that Yap1^CA can still be phosphorylated on non-mutated residues, but this is not sufficient to alter YAP1 nuclear localization (n = embryos).