Micrographs of Xenopus laevis embryos at developmental stages used for iTRAQ measurements (A). Design and workflow of three independent iTRAQ experiments (B). Three experiments were performed. In experiment 1 (E1), two embryos at stage 1, two embryos at stage 5, two embryos at stage 8, and two embryos at stage 11 were separately lysed and digested with trypsin. The first embryo at stage 1 was labeled with the iTRAQ reagent channel 113, the second embryo at stage 1 was labeled with the iTRAQ reagent channel 114, the two embryos at stage 5 were labeled with the iTRAQ reagents channels 115 and 116, the two embryos at stage 8 were labeled with the iTRAQ reagent channels 117 and 118, and the two embryos at stage 11 were labeled with the iTRAQ reagent channels 119 and 121. These labeled peptides were pooled, subjected to strong cation exchange chromatography fractionation, and each fraction was analyzed using reversed-phase liquid chromatography and detection with a Q-Exactive mass spectrometer. Tandem mass spectra were analyzed both to identify the peptide and to quantitate the abundances of each peptide from each of the eight embryos. A similar procedure was performed in experiment 2 (E2), except that the biological duplicates consisted of single embryos taken from stages 1, 5, 13 and 22. Finally, experiment 3 (E3) employed four pools of four embryos, where each pool was taken embryos at stages 1, 8, 13 and 22.

Proteins with significant change of abundance are grouped into one of six clusters according to the changes in expression as a function of developmental stage. Log₂(protein abundance ratio) from experiment 3 (E3) was used for cluster analysis. The number of clusters was fixed at 6. The upper regulation threshold for the log₂ data was 0.26 and lower threshold was −0.32 corresponding to the original ratios of 1.2 and 0.8.

The expression levels of several DNA replication factors and histones change at the mid-blastula transition. (A) Xcdc6 exhibits a marked decline after the mid-blastula transition relative to other replication factors. (B) Levels of Cdc6 protein measured by western blot for control embryos and embryos injected with Cdc6 mRNA (1 ng). (C) Overexpression of Cdc6 triggers increased levels of apoptosis. Apoptosis was detected by staining late blastula (stage 9) embryos with PSS-380 for the fluorescent detection of phosphatidylserine. Left panel is water-injected control embryo; center panel is Cdc6 mRNA (1 ng) injected embryo; right panel is embryo incubated with 0.1 μM staurosporine to induce apoptosis. (D) Levels of maternal histone H1 (H1M/B4) decline while levels of adult H1 and core histones increase. Data from experiments E1 and E3 were used to generate (A) and (D), and the error bars for stage 8 were based on the results from experiments E1 and E3.