The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo. and aPKC may cooperate with heterotrimeric G proteins to regulate microtubule motor protein dynein (Neumuller and Knoblich, 2009; Nguyen-Ngoc et al., 2007; Park and Rose, 2008; Srinivasan et al., 2003; Suzuki and Ohno, 2006). Interestingly, we find here that dynein is usually indeed involved in maintaining apical localization of the nuclei. In conclusion, our results suggest that the extent of Cdx2 and aPKC expression, 481-74-3 IC50 together with microtubule cytoskeleton, affect localization of nuclei in the 8-cell mouse embryo and, consequently, the plane of the cell division (Fig. 4D). In this model, the higher expression of Cdx2, the more intensely aPKC accumulates in the apical region which, in turn, facilitates pulling causes excreted by dyneins on microtubules attached to the nucleus to help sustain its apical localization. Cells with apically located nuclei divide almost always symmetrically. On the other hand, when Cdx2 is usually low, the apically directed pulling force is usually weakened and the nucleus is usually moved by kinesins along microtubules towards the baso-central region of a cell. Such cells with nuclei 481-74-3 IC50 positioned baso-centrally maintain a flexibility regarding the division plane. Effectively all asymmetric divisions take place in cells with nuclei localized towards the basal region but these are still the minority of this group. Our results therefore suggest that apart from the position of the nucleus, other factors influence the cleavage plane. In agreement with this, the effect of misregulating the cell polarity network, for example through dn aPKC, has a greater effect upon the proportion of asymmetric divisions than it does on nuclear positioning. Thus, it is usually important to place the mechanism for nuclear positioning identified here into the context of other factors 481-74-3 IC50 affecting spindle positioning, such as cell polarity itself, to understand how division is usually oriented in the mouse embryo. Materials and methods Animals F1 (C57Bl6xCBA) and Cdx2 oocyte-specific Cre-mediated knock-out (Cdx2KOZp3Cre) (Gao et al., 2009; Kaneda et al., 2009) mouse females and F1, Histone 2B-GFP (H2B-GFP) (Hadjantonakis and Papaioannou, 2004) and heterozygous Cdx2+/? males (Chawengsaksophak et 481-74-3 IC50 al., 1997) were used for the experiments. Animals were maintained in the Animal Facility of Gurdon Institute at 12:12 light cycle and provided with food and water ad libitum. Experiments were conducted in compliance with the University of Cambridge regulations. Embryo collection and culture Females were superovulated with intraperitoneal injection of 7.5?IU of pregnant mare serum gonadotrophin (Intervet) and 48?h later of 7.5?IU of human chorionic gonadotrophin (Intervet). 2-Cell embryos were recovered 42?h later from oviducts into M2 medium and cultured in KSOM medium until 8-cell stage, as described before (Bischoff et al., 2008). In some experiments pre-compacted 8-cell embryos were moved to M2 medium and cultured for FBXW7 8?h (until late G2-phase) with 5?g/ml nocodazole, 2?g/ml cytochalasin Deb, 500?M sodium orthovanadate. Microinjections and live imaging of embryos Constructs encoding Cdx2, dn aPKC, and Gap43 tagged with RFP or GFP were cloned into a pBluescript RN3P vector and mRNA was synthesized from T3 promotor using mMessage mMachine T3 kit (Ambion), as established previously (Zernicka-Goetz et al., 1997). Construct encoding Histone 2B tagged with RFP was cloned into pGEMHE vector and synthesized from T7 481-74-3 IC50 promotor using mMessage mMachine T7 kit (Ambion). mRNAs (0.05?g/l for Cdx2, 0.23?g/l for dn aPKC, 0.34?g/l for Gap43-RFP and Gap43-GFP, 0.05?g/l for H2B-RFP) were injected into one 2-cell embryo blastomere and injected embryos were cultured in KSOM until late 4- or 8-cell stage. In some experiments 8-cell embryos? cells were injected with either rabbit pan-kinesin antibody (Cytoskeleton, 250?g/ml), or control rabbit IgG (250?g/ml). In both cases rhodamine dextran was used as a cell lineage tracer. Embryos were transferred to M2 medium and imaged in 12C15 planes (5C7?m apart) every 15?min for 12?h over the 8C16-cell transition. Imaging was performed on Leica scanning confocal microscope, Zeiss spinning-disc confocal microscope or Deltavision fluorescence microscope, equipped with 37.5?C chambers. Immunostaining Embryos were fixed in 4% PFA (30?min,.