The genome of the basal choradate Ciona intestinalis contains a basic set of genes with less redundancy compared to the vertebrate genome. Extensive EST analyses, cDNA sequencing, and clustering yielded "Ciona intestinalis Gene Collection Release 1," which contains cDNA clones for 13,464 genes, covering nearly 85% of the Ciona mRNA species. This release is ready for use in cDNA cloning, micro/macroarray analysis, and other comprehensive genome-wide analyses for further molecular studies of basal chordates.

Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

A set of 3423 expressed sequence tags derived from the Ciona intestinalis tailbud embryos was categorized into 1213 independent clusters. When compared with DNA Data Bank of Japan database, 502 clusters of them showed significant matches to reported proteins with distinct function, whereas 184 lacked sufficient information to be categorized (including reported proteins with undefined function) and 527 had no significant similarities to known proteins. Sequence similarity analyses of the 502 clusters in relation to the biosynthetic function, as well as the structure of the message population at this stage, demonstrated that 390 of them were associated with functions that many kinds of cells use, 85 with cell-cell communication and 27 with transcription factors and other gene regulatory proteins. All of the 1213 clusters were subjected to whole-mount in situ hybridization to analyze the gene expression profiles at this stage. A total of 387 clusters showed expression specific to a certain tissue or organ; 149 showed epidermis-specific expression; 34 were specific to the nervous system; 29 to endoderm; 112 to mesenchyme; 32 to notochord; and 31 to muscle. Many genes were also specifically expressed in multiple tissues. The study also highlighted characteristic gene expression profiles dependent on the tissues. In addition, several genes showed intriguing expression patterns that have not been reported previously; for example, four genes were expressed specifically in the nerve cord cells and one gene was expressed only in the posterior part of muscle cells. This study provides molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. The sequence information will also be used for further genome scientific approach to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans.

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