Dana Ann A. Tamashiro

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

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.

Aggregated P19 Mouse Embryonal Carcinoma Cells as a Simple In Vitro Model to Study the Molecular Regulations of Mesoderm Formation and Axial Elongation Morphogenesis

Because of their capacity to give rise to various types of cells in vitro, embryonic stem and embryonal carcinoma (EC) cells have been used as convenient models to study the mechanisms of cell differentiation in mammalian embryos. In this study, we explored the mouse P19 EC cell line as an effective tool to investigate the factors that may play essential roles in mesoderm formation and axial elongation morphogenesis. We first demonstrated that aggregated P19 cells not only exhibited gene expression patterns characteristic of mesoderm formation but also displayed elongation morphogenesis with a distinct anterior–posterior body axis as in the embryo. We then showed by RNA interference that these processes were controlled by various regulators of Wnt signaling pathways, namely β‐catenin, Wnt3, Wnt3a, and Wnt5a, in a manner similar to normal embryo development. We further showed by inhibitor treatments that the axial elongation morphogenesis was dependent on the activity of Rho‐associated kinase. Because of the convenience of these experimental manipulations, we propose that P19 cells can be used as a simple and efficient screening tool to assess the potential functions of specific molecules in mesoderm formation and axial elongation morphogenesis. genesis 47:93–106, 2009.

Rho-Associated Kinase Activity Is Required for Proper Morphogenesis of the Inner Cell Mass in the Mouse Blastocyst

ABSTRACT The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

Ectopic expression of mouse Sry interferes with Wnt/β-catenin signaling in mouse embryonal carcinoma cell lines

In mammals, Sry is the master regulator of male sex determination, although how it functions is still unclear. By contrast, female sex determination depends on the action of Rspo1 and Wnt4, the regulators of Wnt/beta-catenin signaling. To seek a possible interaction between male and female sex determination mechanisms, we examined whether Sry affects Wnt/beta-catenin signaling. Using the TOPFLASH reporter system to measure Lef/Tcf-dependent transcriptional activity, we showed that ectopic expression of mouse Sry strongly suppressed Wnt/beta-catenin signaling in mouse embryonal carcinoma and human embryonic kidney cell lines. This inhibition occurred downstream of beta-catenin but upstream of Lef/Tcf, and depended on both the HMG-box and the C-terminal transcriptional activation domain. By contrast, TOPFLASH was not inhibited by human SRY, which apparently lacks a transcriptional activation domain. However, a fusion construct consisting of human SRY attached to the C-terminal domain of mouse Sry was able to inhibit TOPFLASH effectively. Furthermore, Sry constructs carrying point mutations equivalent to those in human sex reversal mutations were less effective in inhibiting Wnt/beta-catenin signaling. Also, we showed that the action of Sry as a transcriptional activator was both necessary and sufficient to inhibit Wnt/beta-catenin signaling, suggesting that the transcriptional targets of Sry are responsible for the inhibition of signaling. Sox9 is a potential transcriptional target of Sry, although quantitative RT-PCR analysis indicates that the expression of Sox9 was not up-regulated by the ectopic expression of mouse Sry in mouse embryonal carcinoma cells. While the present study demonstrates an impact of mouse Sry on Wnt/beta-catenin signaling at an in vitro level, it requires further investigations to assess whether such action also takes place in vivo to regulate male sex determination.

Dual Roles of Oct4 in the Maintenance of Mouse P19 Embryonal Carcinoma Cells: As Negative Regulator of Wnt/β-Catenin Signaling and Competence Provider for Brachyury Induction

Transcription factor Oct4 is expressed in pluripotent cell lineages during mouse development, namely, in inner cell mass (ICM), primitive ectoderm, and primordial germ cells. Functional studies have revealed that Oct4 is essential for the maintenance of pluripotency in inner cell mass and for the survival of primordial germ cells. However, the function of Oct4 in the primitive ectoderm has not been fully explored. In this study, we investigated the role of Oct4 in mouse P19 embryonal carcinoma (EC) cells, which exhibit molecular and developmental properties similar to the primitive ectoderm, as an in vitro model. Knockdown of Oct4 in P19 EC cells upregulated several early mesoderm-specific genes, such as Wnt3, Sp5, and Fgf8, by activating Wnt/β-catenin signaling. Overexpression of Oct4 was sufficient to suppress Wnt/β-catenin signaling through its action as a transcriptional activator. However, Brachyury, a key regulator of early mesoderm development and a known direct target of Wnt/β-catenin signaling, was unable to be upregulated in the absence of Oct4, even with additional activation of Wnt/β-catenin signaling. Microarray analysis revealed that Oct4 positively regulated the expression of Tdgf1, a critical component of Nodal signaling, which was required for the upregulation of Brachyury in response to Wnt/β-catenin signaling in P19 EC cells. We propose a model that Oct4 maintains pluripotency of P19 EC cells through 2 counteracting actions: one is to suppress mesoderm-inducing Wnt/β-catenin signaling, and the other is to provide competence to Brachyury gene to respond to Wnt/β-catenin signaling.

Nkx1-2 is a transcriptional repressor and is essential for the activation of Brachyury in P19 mouse embryonal carcinoma cell.

Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.