Anchoring of maternal RNAs in the oocyte of the clawed frog, Xenopus laevis

grantor: University of TorontoThe body plan of the embryo is established by a polarized source of developmental information in the oocyte. The 'X. laevis' oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including ' Vg1', which may function in body plan specification, and ' Xwnt-11' and 'Xcat-2', which may function in germ cell development. To identify components of the RNA anchoring mechanism, I used manually isolated vegetal cortices (IVC) to assay loss or change in spatial arrangement of RNAs caused by disruption of cortical elements. The role of cytoskeleton in RNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin filaments and cytokeratin filaments, and by using the IVC to assay for RNAs. Treatment of oocytes with cytochalasin B caused clumping of 'Vg1' and 'Xwnt-11' as revealed by 'in situ' hybridization of IVCs, but did not cause their release as confirmed by RT-PCR analysis. These RNA clumps did not match the distribution of actin filament clumps, but they appeared to be similarly distributed as the remnant cytokeratin filaments. Treatment of oocytes with anti-cytokeratin antibody C11 released these RNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. From these results, I concluded that 'Vg1' and 'Xwnt-11 ' are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depended on an intact actin meshwork. Colcemid did not disrupt 'Vg1' and 'Xwnt-11' retention by the IVC, so anchoring of these RNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased 'Vg1 ' and 'Xwnt-11'. The disappearance of these RNAs was mainly due to degradation by ribonuclease activity released from membrane components. However, more 'Vg1' and 'Xwnt-11' were recovered in the supernatant upon treatment of IVCs on ice, which diminished ribonuclease activity. This suggested that a fraction of these RNAs required membranes to be retained in the cortex. Cytochalasin B, C11, colcemid, and Triton X-100 on ice neither released nor degraded 'Xcat-2' mRNA, so no cortical element could be implicated in its anchoring. 'Xcat-2' was also more resistant to endogenous ribonuclease activity compared to 'Vg1 ' and ' Xwnt-11'.Ph.D

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

Polarity-Dependent Distribution of Angiomotin Localizes Hippo Signaling in Preimplantation Embryos

SummaryBackgroundIn preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown.ResultsWe show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway.ConclusionsWe propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates