Embryogenesis in ascidians is known to be of the mosaic type, a property that suggests the presence of cytoplasmic factors in the egg which are responsible for specification of the developmental fates of early blastomeres. Endoderm cells are present in the trunk region of tadpole larvae, and these cells specifically express alkaline phosphatase (AP). Endoderm cells originate exclusively from blastomeres of the vegetal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of endoderm specification, we carried out cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various regions. Initially, presumptive-epidermis blastomeres (blastomeres from the animal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of endoderm cells was monitored by histochemical staining for AP. AP activity was observed only when presumptive-epidermis blastomeres were fused with cytoplasmic fragments from the presumptive-endoderm blastomeres. The results suggest that cytoplasmic factors that promote the initial event of endoderm differentiation (endoderm determinants) are present in endoderm-lineage blastomeres. Next, to examine the presence and localization of endoderm determinants in the egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the presumptive-epidermis blastomeres. The results suggest that endoderm determinants are already present in unfertilized eggs, and that they are segregated by movements of the ooplasm after fertilization. Initially, these determinants move to the vegetal pole of the egg. Then, prior to the first cleavage, their distribution extends in the equatorial direction, namely, to the entire vegetal hemisphere from which future endoderm-lineage blastomeres are formed.
3D structured illumination microscopy of mammalian embryos and spermatozoa