Smith 9724094 The development of a complex organism, such as a vertebrate, from a fertilized egg is a multi-step process involving the coordinated actions of thousands of genes. In elaboration of the adult form from the egg, mechanisms involving both the segregation of cell fate determinants to specific regions of the developing embryo, and inductive interactions, in which cells communicate to determine the developmental fates of neighboring cells, are found. In this project Dr. Smith will seek to isolate genes that act early in chordate development and which are involved in determining the fates of specific tissues. As a model chordate in these studies, he will use the ascidian Ciona intestinalis. Ciona, which is one of the most primitive of the chordates, has many advantages for study of early chordate development. While ascidians retain the basic body plan common to chordates, they have a fixed lineage in early development, they have very few cells at the larvae stage ((2,300), and they have a genome that is only about 5% the size of a vertebrate. To identify genes which are important for early development in Ciona, an expression cloning strategy will be used. Plasmid cDNA libraries will be made from Ciona gastrula stage embryos. In vitro transcripts from the libraries will be microinjected as pools into 4-cell stage Xenopus embryos. Pools with activity will be identified as those that bring about developmental changes to the Xenopus embryos. Single cDNAs with activity will be isolated by sub-dividing the libraries into progressively smaller pools. The identity of genes isolated by this method will be determined by nucleotide sequencing, and the expression patterns will be determined by in situ hybridization to staged Ciona embryos.
