The sequenced genome of the urochordate ascidian Ciona intestinalis contains nearly 2,500 genes that have vertebrate homologues, but their functions are as yet unknown. To identify novel genes involved in early chordates embryogenesis, we previously screened 200 Ciona genes by knockdown experiments using specific morpholino oligonucleotides and found that suppression of the translation of 40 genes caused embryonic defects (Yamada et al. [2003] Development 130:6485-6495). We have since examined an additional 304 genes, that is, screening 504 genes overall, and a total of 111 genes showed morphological defects when gene function was suppressed. We further examined the role of these genes in the differentiation of six major tissues of the embryo: endoderm, muscle, epidermis, neural tissue, mesenchyme, and notochord. Based on the similarity of phenotypes of gene knockdown embryos, genes were categorized into several groups, with the suggestion that the genes within a given group are involved in similar developmental processes. For example, five were shown to be novel genes that are likely involved in beta-catenin-mediated endoderm formation. The type of large-scale screening used is, therefore, a powerful approach to identify novel genes with significant developmental functions, the details of which will be determined in future studies.