KHDC1B Interacts with CPEB Figure 3. Expression proﬁle for KHDC1 proteins. (A) Antibody speciﬁcity was tested by Western blotting. 293T cells were transfected with the indicated plasmids. The pan-KHDC1 antibody recognizes both 1A and 1B. The peptide antibody ( -KHDC1A) is speciﬁc for 1A. (B) KHDC1A is expressed in multiple tissues as detected using the -KHDC1A antibody. The predicated size for KHDC1A is 19.6 kDa. In addition to the full-length form, a 15 kDa isoform of 1A is detected in testis. Tubulin is shown as a loading control. (C) The -KHDC1A antibody detects a 15-kDa isoform in germinal vesicle (GV), germinal vesicle breakdown (GVBD), and meiotic M-phase I (MI) and meiotic M-phase II (MII) stage oocytes. Expression was also detected in early cleavage stage embryos. (D) Immunostaining for pan-KHDC1 in frozen sections taken from adult ovaries. Preantral follicles containing oocytes surrounded by two layers of granulosa cells or antral follicles surrounded by multiple layers of granulosa cells are shown. Staining is either cytoplasmic or nuclear, and arrows indicate the position of the oocyte. The antibody also stains granulosa cells (g) and ovarian stroma (s). An antral follicle processed without primary antibody is shown as a negative control. Scale bar 82 m. (E) Immunostaining for pan-KHDC1 during meiotic maturation and early embryonic development. Staining is shown in germinal vesicle (a), germinal vesicle breakdown (b), and meiotic M-phase II (c) stage oocytes and 1-cell (d and e), 2-cell (f), 4-cell (g), 8-cell (h), 16-cell (i), morula (j), and blastocyst (k) stage embryos. A two-cell embryo processed without primary antibody (l) is shown as a negative control. Arrows indicate perinuclear staining. Scale bar 20 m. phenotype is characteristic of perturbations affecting cell cycle progression, which in Xenopus embryos is controlled by translational regulators (Mendez and Richter, 2001). Em- bryos injected with Khdc1b die shortly after midblastula transition (Figure 5D). Cell division requires coordination of both cytoplasmic and nuclear processes. To see whether KHDC1B expression affects the cytoplasmic or nuclear aspects of division, we examined the distribution of microtubules (Figure 5B) and DNA (DAPI) (Figure 5C) in Khdc1b-injected embryos. The MT cytoskeleton in the cleavage-arrested cells was com- pletely disrupted (Figure 5B). Mitotic asters could be readily identiﬁed in control cells, whereas KHDC1B expressing cells Vol. 21, September 15, 2010 typically contained many small foci of - tubulin staining. Additionally, regions expressing KHDC1B had fewer nuclei indicating arrest of both nuclear division and cytokinesis (Figure 5C). The above phenotypes bear a striking resem- blance to phenotypes resulting from inhibition of the trans- lational regulator, CPEB (Groisman et al., 2000). In Xenopus, CPEB controls cell cycle progression before the midblastula transition. It has been shown to regulate polyA tail length and translation of Cyclin B1 and other target mRNAs in- volved in cell cycle control (Richter, 2007). To test whether KHDC1B overexpression perturbs the cell cycle by interfer- ing with CPEB activity, we performed rescue experiments. The majority of Khdc1b-injected embryos have a cleavage 3141