Ci-Tbx6b and Ci-Tbx6c are key mediators of the maternal effect gene Ci-macho1 in muscle cell differentiation in Ciona intestinalis embryos.

Maternally deposited mRNA encoding the Zic family zinc-finger protein Ci-macho1 is a determinant responsible for muscle cell differentiation in Ciona intestinalis embryos. In a previous study, we identified possible Ci-macho1 downstream genes, which include seven transcription factor genes and seven signaling molecule genes (Yagi, K., Satoh, N., Satou, Y., 2004. Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1. Dev. Biol. 274, 478-489), suggesting complex Ci-macho1 downstream cascades. Here, we show that of the Ci-macho1 downstream genes, only Ci-Tbx6b and Ci-Tbx6c promote ectopic differentiation of muscle cells when misexpressed in non-muscle blastomeres. Overexpression of Ci-Tbx6b or Ci-Tbx6c in Ci-macho1 knockdown embryos is able to compensate for the functional loss of Ci-macho1 and promote differentiation of muscle cells. In addition, we show that knockdown of each of Ci-Tbx6b or Ci-Tbx6c suppresses the initiation of muscle protein gene expression, and both gene products appear to recognize a similar binding sequence. However, later expression of muscle protein genes at the tailbud stage is only reduced in Ci-Tbx6b knockdown embryos and undisturbed in Ci-Tbx6c knockdown embryos. Although ectopic expression or knockdown of Ci-ZicL alone does not affect muscle cell differentiation, simultaneous knockdown of Ci-Tbx6b, Ci-Tbx6c, and Ci-ZicL completely abolishes muscle cell differentiation, as in the case of knockdown of Ci-macho1 and Ci-ZicL. These results strongly suggest that muscle cell differentiation in Ciona embryos is controlled by four key factors: maternal macho1 and zygotic Tbx6b, Tbx6c, and ZicL. The two T-box genes are primary mediators of macho1 function, and cooperation between the zygotically expressed transcription factors is indispensable for muscle cell differentiation in Ciona embryos.