In animal development, the mechanisms by which localized factors and organelles in egg cytoplasm were exactly distributed into each daughter cell are essential for formation of various cell types. During ascidian Halocynthia roretzi embryogenesis, ooplasmic mitochondria were mainly segregated into muscle and neural precursor cells. At the 32-cell stage, localized mitochondria in the B6.2 blastomeres were preferentially distributed into the B7.4 muscle precursors compared with the B7.3 mesenchyme/ notochord precursors. When the B6.2 blastomeres were isolated from the early 32-cell stage embryos and then allowed to divide 2 times of cell division, the resultant partial embryos showed symmetric distribution of mitochondria, and the partial embryos were composed of equal size cells. In normal development, cell fates of the B7.3 blastomere were correlated with the unequal cleavage of B6.2 lineage cells that normally occurs in the next two-cell division stages to produce a large B8.5 mesenchyme and a small B8.6 notochord cell. Mitochondria are distributed asymmetrically in both cells. When embryos were treated with FGF receptor inhibitor SU5402 and MEK inhibitor U0126 between the 32-cell and the early 64-cell stages, the resultant embryos showed equal cleavage pattern and symmetric distribution of mitochondria in daughter cells of the B6.2 blastomeres. However, blocking of Nodal and Notch signaling did not affect the cell division pattern and mitochondrial distribution in the B6.2 lineage blastomeres between the 32-cell and 110-cell stages. Therefore, it is likely that FGF/MEK signaling is involved in asymmetric distribution of mitochondria and unequal cleavage of the B6.2 lineage blastomeres in ascidian embryo.

The mechanisms by which embryo exactly distributes mitochondria into the blastomeres during embryogenesis are one of the important issues in developmental biology. Although the mechanisms has been thought to be important for the proper embryonic development, our understanding has remained limited. In the present study, the distribution of mitochondria was examined in embryos of the ascidian, Halocynthia roretzi, by immunohistochemical staining with three-types of the mitochondria-specific antibodies and vital staining of mitochondria with a fluorescent probe, DiOC2(3). Results of the immunohistochemical staining coincided with that of vital staining, which is able to detect the distribution of mitochondria in cytoplasm of the embryo. Mitochondria was mainly segregated into the B4.1 posterior-vegetal blastomeres at the 8-cell stage. During the next stages, mitochondria was preferentially partitioned into cells of the B-line muscle and the A-line nerve cord precursor compared with each sister cell, endoderm in the 5th cleavage stage, and mesenchyme and notochord in the 6th cleavage stage. However, the mitochondria-rich cytoplasm is divided equally among the blastomeres of the animal hemisphere between the 8-cell and the 64-cell stages. When B6.2 blastomeres were isolated at the early 32-cell stage embryo and cultured in seawater, until control embryos reached the 64-cell stage, pattern of mitochondria distribution was similar to results of the coisolated B7.3 and B7.4 blastomeres from the 64-cell stage embryos. Therefore, it is likely that mitochondria are asymmetrically segregated into the marginal cells in the vegetal hemisphere of the ascidian embryo without cell-cell interaction.