Gene expression during body muscle differentiation in ascidians (Tunicata) and the evolution of muscle in chordates

Ascidians present three types of muscles in their life: striated in the larval tail and heart, and smooth in the post-metamorphic sessile phase. The larval and cardiac muscles have an arrangement of myofilaments like the striated muscle of vertebrates. Instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. A great number of ascidians form colonies constituted of clonal individuals (blastozooids) originated by asexual reproduction. We studied the musculature in the colonial ascidian Botryllus schlosseri (Fig. 1) analysing its organization, differentiation and gene expression during development of blastozooids, beginning from the early bud stage to adult and regression stage. We isolated and characterised two transcripts from colonies that resulted homologous to muscle genes of solitary ascidians: a muscletype actin (BsMA2), a cytoplasmic-type actin (BsCA1) and a troponin T (BsTnT-c). Moreover, we obtained also the genomic sequences coding for BsMA2 and BsCA1. Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. The BsMA2 and BsCA1 genomic sequences were compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our data revealed that intron positions are conserved in ascidians and in the other deuterostomes. We detected the expression of the two genes by in situ hybridization on section (ISH; Fig. 2), in order to follow the muscle development throughout the blastogenetic cycle of B. schlosseri. The ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of BsMA2 and BsTnT-c labels differentiating muscles which appear in the intersiphonal area of young buds. Then, the muscle fibres differentiate into the body-wall, while an intense expression only of BsMA2 marks the myocardium just when it begins contractions. The organization and differentiation of striated cardiac and smooth muscle cells were also investigated at ultrastructural level (Fig. 2) during all the phases of the blastogenetic cycle from early bud to zooid regression.