Ultraviolet-Sensitive Determinants of Gastrulation and Axis Development in the Ascidian Embryo

Abstract

The axes of the ascidian embryo are determined after fertilization during a series of cytoplasmic movements known as ooplasmic segregation. UV irradiation of the vegetal hemisphere of the fertilized Styela egg during ooplasmic segregation prevents gastrulation, axis formation, and brain sensory cell differentiation without affecting subsequent cytoplasmic movements, the early cleavage pattern, or differentiation of muscle, epidermal, and endodermal cells. Proteins synthesized in UV-irradiated (axis-deficient) and normal embryos were compared by 2D-gel electrophoresis and autoradiography. Of 433 polypeptides detected in normal embryos, only about 5% were missing or decreased in labelling intensity in UV-irradiated embryos. The most prominent of these is a 30,000 molecular weight (pI 6.0) polypeptide (p30). Several lines of evidence suggest that p30 may play a role in embryonic axis formation. First, p30 labelling peaks during gastrulation, when the embryonic axis is being established. Second, p30 labelling and axis formation are abolished by the same threshold UV dose. Third, the UV sensitivity period for abolishing p30 labelling and axis formation are both restricted to ooplasmic segregation. Because the chromophore(s) involved in axis determination exhibits absorption characteristics similar to nucleic acids, rather than proteins, and p30 is not detectibly synthesized until after the conclusion of the UV sensitivity period, p30 cannot be the UV sensitive target localized in the vegetal pole region. In vitro translation of egg RNA and subsequent analysis of protein products showed that p30 is encoded by a maternal mRNA. To determine whether UV irradiation inactivates p30 mRNA, RNA from UV-irradiated and normal eggs was translated in a cell-free system and the labelled proteins were compared by gel electrophoresis. The results showed that translation of p30 mRNA, as well as a number of other mRNAs, was abolished in UV-irradiated embryos. These results show that gastrulation and axis formation are controlled by UV-sensitive components transiently localized in the vegetal hemisphere during ooplasmic segregation and that p30 mRNA is a UV target. UV irradiation of the vegetal pole may affect development by inactivating localized mRNAs encoding proteins that function in gastrulation and axis formation.