P99. Paraxial T-box genes, Tbx6 and Tbx1, are required for cranial chondrogenesis and myogenesis

We previously reported that Tbx6, a T-box transcription factor expressed in nascent nonaxial mesoderm, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation in Xenopus laevis . Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly identical to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. In order to clarify the reasons why Tbx6 knockdown affects the development of head and pharyngeal muscles and cartilages, we noticed on Tbx1, another T-box gene expressed in more anterior paraxial structures, and a gene responsible for human DiGeorge syndrome, as a candidate to mediate Tbx6 function. Tbx6 knockdown reduced the expression of Tbx1. Next, Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. Tbx6 expression in Tbx1 knockdown embryos was not reduced at all, but rather expanded in anterior direction, suggesting that Tbx6 is epistatic to Tbx1. This expansion of Tbx6 expression in Tbx1 morphants, when considered together with the fact that there was a gap between the anterior Tbx1-expressing and posterior Tbx6-expressing areas in normal neurula to early tailbud embryos, implys the presence of interaction between the anterior and posterior paraxial mesodermal tissues. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9.