KHDC1B Interacts with CPEB Figure 5. Ectopic expression of KHDC1 proteins perturb Xenopus development. (A) 1 ng of Flag-Khdc1a or Flag-Khdc1b RNA was injected into one cell of two-cell stage Xenopus embryos. Phenotypes were scored at stage 6.5 or at stage 8.5. KHDC1A expression caused a small cell phenotype by stage 6.5 (outlines) and cell death by 8.5. KHDC1B expression caused cleavage arrest (outlines) and eventually cell death after stage 8.5 (see D). Scale bar 500 m. (B) Ectopic expression of KHDC1B altered microtubule distribution. Frozen sections from control or Khdc1b-injected embryos were stained for -tubulin. Control cells have normal mitotic spindles. Khdc1b-injected cells have many small foci of -tubulin. Scale bar 100 m. (C) Ectopic expression of KHDC1B altered the distribution of nuclei. The KHDC1B expressing region marked by Flag staining contains very few nuclei. Dotted lines indicate the edge of the tissue section and the Flag expressing region. Scale bar 250 m. (d-E) mCPEB1 rescues the KHDC1 phenotypes. 1 ng of the indicated mRNAs was injected into one cell of two-cell stage embryos and embryos were cultured to midblastula transition (stage 9 –9.5). (D) Expression of the control KH domain protein ESG1 did not perturb Xenopus development. Expression of KHDC1B by itself caused cell cycle arrest and death by MBT. Coinjection of a control mRNA encoding for luciferase did not rescue the KHDC1B phenotype. However, coinjection of mCpeb1 rescued both the cell cycle arrest phenotype and embryo survival. Expression of mCPEB1 by itself did not perturb Xenopus development. Expression of KHDC1A caused a small cell phenotype and embryonic death. Expression of mCPEB1 partially rescued the KHDC1A phenotype. Scale bar 500 m. (E) Percent of control and injected embryos exhibiting normal development to midblastula transition (MBT). n the total number of embryos counted. Data were collected from three independent experiments, and error bars show SD. Vol. 21, September 15, 2010 3143