Analysis of morphogenetic determinants in ascidian eggs and embryos by cytoplasmic deletion and microinjection

Abstract

The determination of cell fates in ascidian embryos is thought to be mediated primarily by factors localized in the cytoplasm. Morphogenetic factors were studied in ascidian eggs and early cleavage stage embryos by the partial deletion of blastomeres and by microinjection of sperm cells into anucleate, myoplasm-deficient egg fragments. In the first series of experiments, regions of blastomeres comprised of 20–30% of the volume of one blastomere were deleted from two- and four-celled embryos. Most of the embryos exhibited regulative capabilities, in that normal tadpole larvae developed from the operated embryos, irrespective of the deletion site. In a second series of experiments, the expression of endodermal alkaline phosphatase (AP) and muscle acetylcholinesterase (AchE) were tested in myoplasm-deficient egg fragments that were injected with sperm cells. After the unoperated controls had developed into tadpoles, myoplasm-deficient fragments injected with sperm cells were scored for their ability to express AP and AchE activity. Many of the myoplasm-deficient fragments that contained injected sperm cells expressed AP activity, whereas uninjected myoplasm-deficient fragments did not exhibit AP activity. When myoplasm-deficient fragments injected with sperm cells were tested for their ability to express AchE activity, none of the fragments expressed this muscle cell marker. Similar results were obtained using two species, Halocynthia roretzi and Styelaplicata. These results suggest that nuclear events are required for the expression of AP and that the myoplasmic region of the fertilized egg contains muscle cell determinants.