Otx2 is expressed in the mesencephalon and prosencephalon, and Gbx2 is expressed in the rhombencephalon around stage 10. Loss-of-function studies of these genes in mice have revealed that Otx2 is indispensable for the development of the anterior brain segment, and that Gbx2 is required for the development of the isthmus. We carried out gain-of-function experiments of these genes in chick embryos with a newly developed gene transfer system, in ovo electroporation. When Otx2 was ectopically expressed caudally beyond the midbrain-hindbrain boundary (MHB), the alar plate of the metencephalon differentiated into the optic tectum instead of differentiating into the cerebellum. On the other hand, when Gbx2 was ectopically expressed at the mesencephalon, the caudal limit of the tectum shifted rostrally. We looked at the effects of misexpression on the isthmus- and tectum-related molecules. Otx2 and Gbx2 interacted to repress each other's expression. Ectopic Otx2 and Gbx2 repressed endogenous expression of Fgf8 in the isthmus, but induced Fgf8 expression at the interface between Otx2 and Gbx2 expression. Thus, it is suggested that interaction between Otx2 and Gbx2 determines the site of Fgf8 expression and the posterior limit of the tectum.

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The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon-intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates.

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