Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2-3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A)+ RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

The spatial distribution of maternal mRNA is determined by a cortical cytoskeletal domain in Chaetopterus eggs

Messenger RNA molecules are localized in the cortical region of eggs and unevenly segregated to the embryonic cells during early development of the annelid Chaetopterus. The egg cortex is enriched in two organelles, ectoplasmic spherules and associated structures, which are similar in appearance to nuage. The physical basis of cortical mRNA localization was examined in stratified eggs and in eggs extracted with the nonionic detergent Nonidet P-40 (NP-40). The cortical organelles were displaced to the most centrifugal zone of stratified eggs. In situ hybridization with poly(U) or cloned DNA probes showed that a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was also displaced to the centrifugal zone. Extraction with NP-40 revealed a detergent-insoluble cytoskeletal domain (CD) in the egg cortex which contained the remnants of ectoplasmic spherules and nuage embedded in a fibrous network. Although most of the total protein and RNA was extracted by NP-40, a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was retained in the CD. In situ hybridization of stratified eggs extracted with NP-40 indicated that the CD, with its associated organelles and mRNA molecules, is displaced to the centrifugal zone as a unit. The results suggest that the tenacious association of mRNA molecules with the cortical CD may be responsible for maternal mRNA localization during early development.     

Spatial distribution of messenger RNA in the cytoskeletal framework of ascidian eggs

Maternal poly(A)⁺RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)⁺RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)⁺RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.     

The location of maternal mRNA in eggs and embryos

Recent studies have shown that some maternal mRNAs are localized in specific cytoplasmic regions of eggs and embryos and are rearranged in concert with the cytoplasmic movements that fix the embryonic axes. The localization and ooplasmic segregation of mRNA molecules may be mediated by their association with specific egg cytoskeletal domains.