Regulation of the muscle-specific expression and function of an ascidian T-box gene, As-T2

The Tbx6 T-box genes are expressed in somite precursor cells of vertebrate embryos and are essential for the differentiation of paraxial mesoderm. However, it is unclear how spatial regulation of the gene expression is controlled and how the genes function to promote muscle differentiation. The Tbx6-related gene As-T2 of the ascidian Halocynthia roretzi is first expressed very transiently in endodermal cells around the 32-∼44-cell stage, is then expressed distinctly and continuously in muscle precursor cells, and later in epidermal cells situated in the distal tip region of the elongating tail. We now show that inhibition of As-T2-mediated transcriptional activation by microinjection of As-T2/EnR into one-cell embryos resulted in suppression of the expression of the muscle-specific actin gene (HrMA4) and myosin heavy chain gene (HrMHC), but the injection did not affect the differentiation of endodermal cells or tail tip cells, suggesting that the primary function of As-T2 is associated with muscle cell differentiation. The 5′ flanking region of As-T2 contains two promoter modules that regulate its specific expression: a distal module that responsible for its specific expression in the tail, and a proximal module required for its muscle-specific expression. Around the proximal module, there are two putative T protein-binding motifs (TTCACACTT). Co-injection of an As-T2/lacZ construct with or without the T-binding motifs together with As-T2 mRNA revealed that these motifs are essential for autoregulatory activation of the gene itself. In addition, we found that the minimal promoter regions of HrMA4 and HrMHC contain T-binding motifs. Co-injection of HrMA4/lacZ or HrMHC/lacZ containing the T-binding motifs along with As-T2 mRNA revealed that As-T2 protein binds to these motifs to upregulate the gene activity. Taking into account the recent finding of maternal molecules for muscle differentiation, we propose a model for a genetic cascade that includes As-T2 as a regulator of muscle cell differentiation in the ascidian embryo.