This project will investigate the egg cytoplasmic determinants in the Ascidian embryo that appear to regulate various of the histospecific features of larval tissue differentiation. Particular emphasis will be placed on a reinvestigation of the muscle cell lineages and on a detailed description of simple muscle-specific characters that respond to control by the lineage-segregated determinant, notably a distinctive muscle acetylcholinesterase, other muscle proteins (myosin and tropomyosin), and simpler ultrastructural features of muscle development (myofilaments and myofibrils). The long-term goals of the project are in isolating the informational agent which influences or selects the pathway to muscle development, determining its chemical properties, and probing its mechanism of action. Microinjection and microsurgical methods will be used with embryos of Ciona intestinalis and Ascidia ceratodes to establish a biological assay for detecting activity of the determinant. Development of acetylcholinesterase and other specific muscle characters after microinjection of myoplasm into non-muscle lineage blastomeres will form the basis of the assay. The assay will be used to monitor the isolation, fractionation, and identification of determinant obtained from the unfertilized egg. Other procedures based on nuclear transplantation and combinations of early and later cytoplasms will provide additional means of assaying some of the temporal and functional properties of the determinant. To evaluate when muscle genes are first expressed (as functional mRNA), and thereby when determinants might first be acting, specific antisera against muscle proteins will be used to identify translational products in the Xenopus oocyte and rabbit reticulocyte lysate systems. These ascidian cytoplasmic factors that act to select and stabilize specific genetic pathways of phenotypic expression are examples of a precocious use in embryogenesis of the same gene regulatory elements that ordinarily function somewhat later in the development of all species, and which have not been easily accessible to study and isolation in higher organisms. Discoveries about the nature and function of egg cytoplasmic determinants in mosaic embryos have, therefore, obvious potential for contributing to an understanding of how gene activity is initially directed in specialized cells.
