Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates.

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