SummaryThe cytoplasmic tyrosine kinase Yes has previously been shown to have an important role in maintaining mouse and human embryonic stem (ES) self-renewal through an unknown pathway downstream of leukemia inhibitory factor (LIF) and one or more factors in serum. Here, we show that TEAD2 and its transcriptional co-activator, the Yes-associated protein YAP, co-operate in a signaling pathway downstream of Yes. We show that YAP, TEAD2 and Yes are highly expressed in self-renewing ES cells, are activated by LIF and serum, and are downregulated when cells are induced to differentiate. We also demonstrate that kinase-active Yes binds and phosphorylates YAP, and activates YAP-TEAD2-dependent transcription. We found that TEAD2 associates directly with the Oct-3/4 promoter. Moreover, activation of the Yes pathway induced activity of the Oct-3/4 and Nanog promoters, whereas suppression of this pathway inhibited promoter activity. Nanog, in turn, suppressed TEAD2-dependent promoter activity, whereas siRNA-mediated knockdown of Nanog induced it, suggesting a negative regulatory feedback loop. Episomal supertransfection of cells with inhibitory TEAD2-EnR induced endodermal differentiation, which suggests that this pathway is necessary for ES cell maintenance.

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.

To investigate the long-term effects of developmental exposure to methylmercury (MeHg), pregnant mice were exposed to at 0.5 mg MeHg/kg/day via drinking water from gestational day 7 until day 7 after delivery. The behavior of offspring was monitored at 5-15 and 26-36 weeks of age using an automated system (IntelliCage) designed for continuous long-term recording of the home cage behavior in social groups and complex analysis of basic activities and learning. In addition, spontaneous locomotion, motor coordination on the accelerating rotarod, spatial learning in Morris water maze, and depression-like behavior in forced swimming test were also studied. The analysis of behavior performed in the IntelliCage without social deprivation occurred to be more sensitive in detecting alterations in activity and learning paradigms. We found normal motor function but decreased exploratory activity in MeHg-exposed male mice, especially at young age. Learning disturbances observed in MeHg-exposed male animals suggest reference memory impairment. Interestingly, the forced swimming test revealed a predisposition to depressive-like behavior in the MeHg-exposed male offspring. This study provides novel evidence that the developmental exposure to MeHg can affect not only cognitive functions but also motivation-driven behaviors.

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3r deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53-and Chk2-related proteins.

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.