S3 D, arrows; Hendershott and Vale, 2014; Jiang et al., 2014). localization. Depletion of CLAMP also affects the polarized business of MTs. We hypothesize that CLAMP facilitates the establishment of cell polarity and promotes the asymmetric build up of MTs downstream of the establishment of appropriate PCP. Introduction The ability of AMG 837 sodium salt cells to coordinately polarize across the plane of the cells requires both cellCcell signaling through the planar cell polarity (PCP) pathway that occurs at the site of cellCcell contacts and intracellular integration of that transmission via cytoplasmic changes to the cytoskeleton. The fundamental aspects of PCP signaling are conserved throughout development, and most of the core components were originally found out in = 43), MOCMO (= 42) and WTCMO (= 82) cells in CLAMP morphant mosaic cells. (C and D) Quantitative analysis (D) of the angle of cell division (representative image, C) measuring the CSD relative to the A-P axis in AMG 837 sodium salt control MO (> 500 cells from seven embryos), CLAMP MO (> 300 cells from seven embryos) and Vangl2 MO cells (> 700 cells from six embryos). In both B and D, error bars represent the SD, and p-values represent the test (two-tailed, type 2). In all images, posterior is definitely to the right. Bars, 5 m. Results and AMG 837 sodium salt conversation Immunostaining having a CLAMP-specific mAb shows that CLAMP weakly localizes to the MT network and enriches at sites of improved MT concentration (e.g., midbodies, centrioles, and cilia; Werner et al., 2014). Importantly, CLAMP also enriches in the apical cell membrane at the site of cellCcell contacts (Fig. 1 A). To test whether this enrichment at cell boundaries is specific, we generated mosaic embryos in which cells designated with blue fluorescent protein contained a previously validated CLAMP morpholino (MO; Werner et al., 2014). Immunofluorescence confirms that CLAMP staining in CLAMP morphant cells is largely absent (Fig. 1, A and B). We quantified this loss specifically at cell contacts by rating the fluorescent intensity of CLAMP relative to the limited junction marker ZO-1 at apical junctions and found a >90% decrease in CLAMP staining at MOCMO boundaries compared with WTCWT boundaries (Fig. 1 B; P < 0.0005). More importantly, when we performed this analysis between WTCMO boundaries, we also saw a significant loss of CLAMP staining, indicating a nonCcell-autonomous effect where loss of membrane-associated CLAMP in one cell prospects to a loss of membrane-associated CLAMP in its neighbor (Fig. 1 B; P < 0.0005). CLAMP has been previously implicated in the radial intercalation of MCCs and ionocytes, yet antibody (Ab) staining reveals that it is indicated throughout all cells of the epithelium. A high dose of CLAMP MO (10 ng in each blastomere in the 4-cell stage) causes early embryonic lethality around stage 14 before MCC differentiation. Consistent with this, our efforts at CRISPR-mediated genome editing also resulted in a total loss of viability, suggesting a broad developmental part for CLAMP. Importantly, targeted injections of MOs into a subset of blastomeres (1 cell in the 4-cell AMG 837 sodium salt stage) generates mosaic embryos that show a much higher survival rate and allow for the assessment of WT and morphant cells in the same embryo (Werner and Mitchell, 2013). In fact, mosaic CLAMP morphant embryos often show an overall stunted and curved growth reminiscent of PCP-mediated convergent extension defects (Wallingford et al., 2002). We observe that during embryo elongation (phases 16C28), the Ets2 majority of mitotic cells align their spindles along the AMG 837 sodium salt anteriorCposterior (A-P) axis having a circular SD (CSD) of 27 (Fig. 1, C and D). Similar to what has been observed in additional elongating cells, we observe that spindle.